authors@Fred Hutch

Zubair, Niha

November 15, 2015 SCIENCE SPOTLIGHT 
 
1 Volume 5, Issue 11 | Fred Hutchinson Cancer Research Center 
 
In flux? Genetics modifies the effect of reflux on 
Barrett's esophagus risk 
November 15, 2015 
     N Zubair 
 
 
 
 
 
Plot of odds ratios for Barrett’s 
esophagus for four strata defined by 
rs2687201 genotype (GG vs. TT, GT) 
and history of gastroesophageal reflux 
disease (GERD) (no vs. yes). The red 
line shows the odds ratios for individuals 
homozygous for the major allele (GG) 
and the blue line shows the odds ratios 
for individuals with at least one copy of 
the minor allele (TT, GT). The stratum 
GG and no GERD is the referent group. 
 
Image provided by Dr. James Dai 
 
 
 
Esophageal adenocarcinoma (EA) is a rare but lethal disease. The majority of EA cases arise from 
Barrett’s esophagus (BE), a condition where the tissue in the tube connecting the mouth and 
stomach (esophagus) is displaced by tissue more akin to the intestinal lining. BE is most often 
diagnosed in those who have long-term gastroesophageal reflux disease (GERD).  In GERD, 
contents from the stomach wash back up into the esophagus, damaging esophageal tissue. As the 
esophagus tries to heal itself, the damaged cells are replaced by the type of cells found in BE. 
However, some patients with BE have never experienced GERD, thus it is unclear what causes BE 
in these individuals. 
BE and EA share many epidemiological risk factors including, European ancestry, male gender, 
obesity, tobacco smoking, and GERD. Not surprisingly, the two diseases share a large proportion of 
genetic heritability: The Barrett’s and Esophageal Adenocarcinoma Consortium (BEACON) 
November 15, 2015 SCIENCE SPOTLIGHT 
 
2 Volume 5, Issue 11 | Fred Hutchinson Cancer Research Center 
 
conducted a genome-wide association study (GWAS) and reported that the risk of BE and EA is 
influenced by many germline variants of small effect.  
Understanding the interplay between genetic susceptibility and epidemiologic risk factors can 
provide insight into disease pathogenesis and etiology. Unfortunately, the statistical power to detect 
genome-wide gene-environment interactions is typically low because of the high variability in 
interaction estimates and the p-value correction needed for genome-wide testing. To address this 
issue, Drs. James Dai, Thomas Vaughan, and colleagues (Public Health Sciences, Vaccine and 
Infectious Disease, and Human Biology Divisions) applied an alternative approach by focusing the 
search for gene-environment interactions to genetic loci previously found to significantly associate 
with BE and EA. Here the premise is if a genetic variant interacts with an exposure, the marginal 
association of this variant (the association regardless of exposure levels) will likely manifest. This 
approach provides a benefit in statistical power when compared to an agnostic genome-wide 
interaction search.  
The authors recently presented their work in Cancer Epidemiology, Biomarkers & Prevention. Using 
data from the BEACON consortium (2,416 BE patients, 1,516 EA patients, and 2,187 controls), the 
authors examined gene-environment interactions of seven SNPs previously shown to associate with 
BE or EA. The environmental risk factors investigated include body-mass index (BMI), cigarette 
smoking, and GERD symptoms. Logistic regression models were used to estimate odds ratios for 
these risk factors stratified by SNP genotype, separately for BE and EA. The authors discovered 
rs2687201 (G/T, major/minor plus strand allele), which lies on chromosome 3 near the FOXP1 gene, 
modified the association between GERD and BE risk.  The presence of at least one minor allele of 
rs2687201 decreased the magnitude of BE risk associated with GERD: odds ratios (95% confidence 
intervals) for GERD among participants with 0, 1, or 2 copies of the minor allele were 6.17 (4.91, 
7.56), 3.56 (2.85, 4.44), and 3.97 (2.47, 6.37), respectively. While the interactions of this SNP with 
GERD on the risk of EA were not statistically significant, the patterns of the odds ratios among the 
genotype groups were similar to those for BE. No other SNP-environment pairs displayed 
statistically significant interactions.  
Next, the authors investigated the interactions between the three environmental risk factors and 
rs2687201 simultaneously. Here the p-value for the interaction with GERD remained nominally 
statistically significant, implying that the newly discovered interaction between the SNP and GERD 
appears to not be confounded by BMI and cigarette smoking.   
Lastly, the authors examined the odds ratios for four strata defined by rs2687201 genotype and 
GERD status; those homozygous for the major allele and without GERD comprised the reference 
November 15, 2015 SCIENCE SPOTLIGHT 
 
3 Volume 5, Issue 11 | Fred Hutchinson Cancer Research Center 
 
group. In the absence of GERD, having at least one minor allele elevated the risk for BE. In the 
presence of GERD, the risk of BE substantially increased (regardless of genotype group), but those 
with at least one minor allele had a smaller BE risk compared to those homozygous for the major 
allele. 
According to Dr. Dai, "the risk association of reflux symptoms and Barrett’s esophagus is modified by 
rs2687201, a known susceptibility locus that lies approximately 75 kilo bases downstream of the 
transcription factor gene FOXP1.  This novel gene– exposure interaction discovered in this study 
provides new insights into the etiology of esophageal adenocarcinoma." FOXP1 encodes a member 
of the Forkhead box (FOX) family of transcription factors, which function as versatile regulators of a 
wide range of biological processes, including development and cancer. Knockout studies in mice 
demonstrated that FOXP1 and FOXP2 cooperatively regulate lung and esophageal development, 
while human FOXP1 was first identified as a candidate tumor suppressor gene. 
Ongoing and future work include expanding beyond known genetic loci; Dr. Dai elaborates, 
"genome-wide testing for gene-exposure interactions for Barrett’s esophagus and esophageal 
adenocarcinoma will reveal more putative loci that modify the risk of known exposures." 
Additional Fred Hutch investigators contributing to this project were Dr. Jean de Dieu Tapsoba, Dr. 
Matthew F. Baus, Lynn E. Onstad, and Dr. Brian J. Reid.  
Dai JY, Tapsoba JdD, Buas MF, Onstad LE, Levine DM, Risch HA, Chow W-H, Bernstein L, Ye W, 
Lagergren J, Bird NC, Corley DA, Shaheen NJ, Wu AH, Reid BJ, Hardie LJ, Whiteman DC, Vaughan 
TL. 2015. A newly identified susceptibility locus near FOXP1 modifies the association of 
gastroesophageal reflux with Barrett's esophagus. Cancer Epidemiol Biomarkers Prev. 24(11):1739-
47.  doi: 10.1158/1055-9965.EPI-15-0507. 
Funding for this study was provided by the National Institutes of Health and by the National Health 
and Medical Research Council of Australia. 
 
 


English

In flux? Genetics modifies the effect of reflux on Barrett's esophagus risk

Koch, Lori

December 20, 2015 SCIENCE SPOTLIGHT 
 
1 Volume 5, Issue 12 | Fred Hutchinson Cancer Research Center 
 
Cleaning out the membrane each day 
December 20, 2015 
     L Koch 
 
 
 
 
Model of heavy isotope labeling 
strategies for monitoring membrane 
lipid dynamics. 
 
Image provided by Dr. Carissa Olsen. 
 
 
 
 
Cell membranes are made up of a lipid bilayer that is constantly changing due to the flux of material 
in and out of the cell.  The staggering variety of lipid species that make up the membrane must be 
constantly maintained in order for the cell to respond to environmental cues or simply grow and 
divide normally.  Aberrant membrane composition is observed in numerous diseases such as 
neurodegenerative diseases and cancer.  Understanding membrane composition and its regulation 
is an important step toward developing effective therapeutics to target and penetrate the abnormal 
membrane of cancer cells. 
There are challenges to studying lipid dynamics in living organisms. The membrane lipids are 
tremendously diverse and they are not genetically encoded.  Therefore, the ideal way to investigate 
how a membrane is maintained over time is to manipulate a relatively simple model organism such 
as the worm, Caenorhabditis elegans.  Another feature of membrane lipids is that they are in 
constant flux; however, there are no established methods to quantify the dynamics of each individual 
population. Researchers in the lab of Dr. Carissa Olsen (Basic Sciences Division) are able to track 
lipid dynamics in great detail using a method they developed in which worms are fed a diet enriched 
for heavy carbon, 
13
C, and then, following a defined growth period, lipids are isolated for 
identification by mass-spectrometry.  Previous work from the lab has demonstrated that the 
13
C label 
December 20, 2015 SCIENCE SPOTLIGHT 
 
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can be used to detect all major species of fatty acids, the tails of lipid molecules. In recent work, 
published in PLoS One, they have extended their analysis of the lipidome—all of the lipids in the 
cell—and outline a new method that uses heavy nitrogen (
15
N) to track intact phospholipids.  
Using heavy carbon labeling and gas chromatography-mass spectrometry (GS-MS), the authors 
were able to quantitatively track how much of each lipid species in the membrane is replaced over 
time.  The rates they measured for each fatty acid predicted that the majority of the fatty acids in the 
membrane are replaced within twenty-four hours. They found good agreement between their 
predictions and the actual measurements of replenishment for most fatty acid species, however 
some fatty acids were not completely replaced in twenty-four hours.  This demonstrates that some 
fatty acids are protected, either actively or inactively, from removal.  
Fatty acid tails are found in polar phospholipids that make up the bilayer and are also found in 
neutral fat-storage lipids such as the common triacylglycerols.  In order to track whether these two 
groups of lipids have different dynamics, the authors physically separated them before analysis of 
each group by GC-MS.  Strikingly, they found that phospholipid maintenance consumes more 
dietary resources than neutral lipids, which are, on average, replaced roughly half as quickly.  This 
observation underscores the importance of preserving membranes over maintaining fat stores in 
adult animals.   
Labeled carbon can come from two sources, either the fully labeled fatty acids from the diet or from 
de novo synthesis, which generates fatty acids with a mixture of labeled to unlabeled carbon.  After 
mathematically separating these two groups during their analysis, the authors found that in general 
most of the new fatty acids incorporated into the membrane come directly from dietary sources, 
although synthesis did account for a significant portion of the population.   
Because the fatty acids in the cell membrane are attached to a phospho-glycerol backbone, directly 
measuring intact phospholipids rather than their isolated fatty acid tails would give a better picture of 
membrane dynamics.  In order to do this, the authors turned to high-performance liquid 
chromatography (HPLC) and mass spectrometry.  The substantial amount of variation in 
phospholipid species prompted the researchers to label only a single atom, the nitrogen in the polar 
head group with a heavy isotope, 
15
N.  They analyzed the most common phospholipid classes in the 
worm membrane, phosphatidylcholine (PtdCho) and phosphotidylethanolamine (PtdEtn), and found 
that PtdEtn phospholipids are exchanged nearly three times the rate of the PtdCho group.  This 
disparity in turnover rates highlights the importance of monitoring the dynamics of individual 
populations and how they are differentially regulated.  Indeed, the methods put forth in this research 
December 20, 2015 SCIENCE SPOTLIGHT 
 
3 Volume 5, Issue 12 | Fred Hutchinson Cancer Research Center 
 
provide a new framework for beginning to dissect the genetics and biochemistry of membrane 
dynamics. 
Dancy BC*, Chen SW*, Drechsler R, Gafken PR, Olsen CP.  2015.  “
13
C- and 
15
N-Labeling 
strategies combined with mass spectrometry comprehensively quantify phospholipid dynamics in C. 
elegans.  PLoS One.  10(11): e0141850. 
*These authors contributed equally to this work. 
This research was funded by the National Institutes of Health and Fred Hutch.  
 
 
 


Cleaning out the membrane each day

Neves, Alexandre

April 20, 2015 SCIENCE SPOTLIGHT 
 
1 Volume 5, Issue 4 | Fred Hutchinson Cancer Research Center 
 
Cyclic Patterns of Inflammatory Cytokines in HIV-
Infected Women 
April 20, 2015 
    A Neves 
Because the major route to HIV infection in women is heterosexual transmission, understanding HIV 
replication in the female genital tract (FGT) is critical, and could have implications for HIV prevention. 
Hormonal changes that occur during the menstrual cycle (MC) could have significant impacts on 
mucosal immunity and HIV shedding, but the mechanism is poorly understood. Cytokines are a 
diverse group of small proteins that act as important signaling molecules in various organ systems, 
particularly the immune system. Accordingly, cytokines are known to involved in the etiology of many 
diseases, including HIV infection, yet the complexity of cytokine kinetics during HIV infection is only 
beginning to be elucidated. 
Previous studies that examined FGT cytokine levels at weekly intervals found elevated levels of 
proinflammatory cytokines during the MC that correlated with HIV shedding. However, it has 
remained unclear whether shedding was regulated by the observed cyclic pattern of cytokines, or by 
the MC itself. A new Fred Hutch study led by former graduate student Dr. Valerie Cortez in the lab of 
Dr. Julie Overbaugh (Human Biology Division), published in Open Forum Infectious Diseases, 
confirmed and extended these observations by examining the daily levels of 26 vaginal cytokines in 
15 women infected with HIV-1 from Mombasa, Kenya. 
The women enrolled in the study attended daily clinics for a full cycle (median of 27 days) and 
cytokine concentrations were obtained from vaginal swabs. The data points were centered around 
the surge of luteinizing hormone (LH). The majority of cytokines exhibited a common pattern; they 
decreased during the follicular phase and subsequently increased steadily during the luteal phase, 
consistent with previous studies of mucosal immunity. After controlling for behavioral factors and 
coexisting sexually transmitted infections, levels of macrophage inflammatory protein (MIP)-1α, 
interleukin (IL)-6, tumor necrosis factor (TNF)-α, interferon (IFN)-α2 and IL-7 were all found to be 
significantly linked to cervical, but not vaginal, HIV shedding. However, this association no longer 
held up after controlling for the number of days that had elapsed since the LH surge. Next, the 
authors used linear mixed-effect models to evaluate the connection between hormone levels and 
cytokine concentrations, and found that progesterone positively correlated with IL-1α and inversely 
correlated with granulocyte colony stimulating factor (G-CSF) and monocyte chemoattractant protein 
(MCP)-1. No significant correlations were found when estrogen was queried. 
April 20, 2015 SCIENCE SPOTLIGHT 
 
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Overall, daily examination of cytokine levels in the FGT of women with chronic HIV-1 infections 
enabled the researchers to unmask several cytokines with cyclical patterns. This study also 
demonstrated that the connection between proinflammatory cytokines and viral shedding is likely to 
be mediated by the hormonal changes that drive the MC. "Our findings highlight the need for future 
longitudinal studies to further define the effects of hormonal changes on the complex immune 
environment in the female genital tract in women, irrespective of HIV status," concluded Dr. Cortez. 
Cortez V, Odem-Davis K, Lehman DA, Mabuka J, Overbaugh J. 2014. Quotidian changes of genital 
tract cytokines in human immunodeficiency virus-1-infected women during the menstrual 
cycle. Open forum infectious diseases, 1(1), ofu002. 
 
Image provided by Dr. Dara Lehman 
Pattern of cytokine and chemokine levels across the menstrual cycle, centered on the luteinizing hormone (LH) 
surge. The graphs show log10 concentrations of cytokines (pg/ml) that were significantly associated with days since 
the luteinizing hormone surge, after adjusting for menstruation in a linear mixed effect model. Each of the grayed 
lines represents one participant, with the dark bolded line denoting the averaged trend across the cycle. Hormonal 
changes during the menstrual cycle are shown in the final panel for reference. 


Cyclic Patterns of Inflammatory Cytokines in HIV-Infected Women

October 19, 2015 SCIENCE SPOTLIGHT 
 
1 Volume 5, Issue 10 | Fred Hutchinson Cancer Research Center 
 
Biomarker candidates for breast cancer found 
using pre-clinical plasma 
October 19, 2015 
     N Zubair 
 
 
 
High-throughput antibody array scan. 
Image provided by Dr. Jung-hyun Rho 
 
 
 
 
Mammography has become a widely used screening tool for breast cancer. Some studies indicate 
that routine mammographic screening reduces breast cancer mortality by 30% among women 50-69 
years of age; however the magnitude of this mortality benefit remains controversial. Under current 
guidelines, age and sex remain the sole factors used in determining whether or not a mammogram 
screening is recommended. This results in millions of healthy women being frequently imaged and 
biopsied, while other women with cancer falsely identified as cancer-free. More refined methods of 
breast cancer risk assessment that integrate clinical parameters could tailor screening 
recommendations; to-date, no clinically useful blood-based biomarkers have been identified for early 
breast cancer diagnosis. Drs. Matthew Buas, Christopher Li, and colleagues in the Public Health 
Sciences Division sought to detect candidate biomarkers for estrogen receptor positive/progesterone 
receptor positive (ER+/PR+) invasive ductal carcinoma, a subtype that accounts for ~45% of 
invasive breast cancer diagnoses in the U.S. As recently reported in Breast Cancer Research and 
Treatment, the authors, using pre-clinical plasma specimens with a customized antibody-array, 
identified a pool of novel candidate protein biomarkers for ER+/PR+ invasive ductal carcinoma; such 
biomarkers, if validated in independent populations, could be of use in selecting subsets of women 
for increased mammography screening relative to current guidelines.    
 
To identify novel protein biomarkers for ER+/PR+ invasive ductal carcinoma, the researchers 
conducted a nested case-control study within the Women's Health Initiative observational study, a 
October 19, 2015 SCIENCE SPOTLIGHT 
 
2 Volume 5, Issue 10 | Fred Hutchinson Cancer Research Center 
 
prospective cohort of over 93,000 post-menopausal women in the U.S. For this study, pre-clinical 
plasma specimens, collected up to 12.5 months before diagnosis from 121 breast cancer cases and 
121 matched controls were available; matching covariates included: age, race/ethnicity, body mass 
index, and hormone replacement therapy use. The researchers divided cases and controls equally 
into training and testing sets and interrogated their plasma samples using a customized antibody 
array. Dr. Buas elaborates, "this study made use of an in-house custom-designed antibody array 
platform developed by Dr. Paul Lampe and colleagues at the Fred Hutch, which enables high-
throughput parallel assessment of >2,000 selected plasma proteins. The platform has evolved over 
the last ~8 years and has been successively refined across multiple generations of array fabr ication 
and development. Several papers have been published describing the platform and its 
implementation in biomarker discovery studies." 
 
When comparing cases to their matched controls, the researchers found statistically significant 
mean differences in signal intensity for 37 distinct proteins; these included both known and novel 
proteins in breast cancer pathogenesis. Four markers remained significant after correcting for 
multiple testing: CSF2, RYBP, TFRC, ITGB4. The last two of these markers have been the focus of 
previous breast cancer research. Studies have linked elevated TFRC protein expression in ER+ 
tumors to higher clinical grade, increased proliferative activity, and worse prognosis. Interestingly, 
this study observed lower levels of TFRC protein in the pre-clinical plasma of cases relative to 
controls; it remains uncertain whether reduced levels of circulating TFRC reflect decreased 
abundance of cellular TFRC, particularly in breast tissue. In contrast to TFRC, the other marker 
previously reported, ITGB4, was elevated in the plasma of cases relative to controls. This marker 
has been linked to increased breast tumor size, nuclear grade, and breast cancer progression.  
 
The researchers next determined whether data from The Cancer Genome Atlas (TCGA) might 
provide further support for any of the markers identified. Of the 37 candidate proteins identified, 14 
exhibited significantly altered changes in RNA expression, when comparing breast tumor versus 
normal tissue.  
 
To assess the utility of combining multiple candidate biomarkers into a composite marker panel, the 
researchers conducted multivariate modeling. The mean area under the curve (AUC) of the models 
built using the 39 antibodies was 0.75 with an estimated sensitivity (true positive rate) of ~30% at 
95% specificity (true negative rate). According to the authors, a marker panel achieving such a 
sensitivity and specificity "would have the potential to capture a significant number of additional 
women who are most likely to benefit from mammography, without flooding the system with those 
least likely to need imaging." 
October 19, 2015 SCIENCE SPOTLIGHT 
 
3 Volume 5, Issue 10 | Fred Hutchinson Cancer Research Center 
 
 
Breast cancer represents a heterogeneous disease with multiple sub-types, defined by distinct 
histological and molecular features. Dr. Buas describes, "by analyzing plasma isolated from women 
who later went on to develop breast cancer (or did not), we could identify proteins that differed in 
abundance prior to breast cancer diagnosis. These marker candidates require external validation in 
independent study populations, but may hold potential clinical value down the road, for applications 
related to early detection or risk stratification."  
 
Additional Fred Hutch investigators contributing to this project were Dr. Jung-hyun Rho, Xiaoyu Chai, 
Yuzheng Zhang, and Dr. Paul D. Lampe. 
 
Citation:  
Buas MF, Rho J-H, Chai X, Zhang Y, Lampe PD, Li CI. 2015. Candidate early detection protein 
biomarkers for ER+/PR+ invasive ductal breast carcinoma identified using pre-clinical plasma from 
the WHI observational study. Breast Cancer Res Treat. 153(2):445-54. 
 
Funding for this study was provided by the National Cancer Institute. 
 


Biomarker candidates for breast cancer found using pre-clinical plasma

October 19, 2015 SCIENCE SPOTLIGHT 
 
1 Volume 5, Issue 10 | Fred Hutchinson Cancer Research Center 
 
It takes ten to tango: the structure of 
transcription factor TFIIH 
October 19, 2015 
     L Koch 
 
Workflow for determining the structure of the 
transcription factor TFIIH. 
 
Image provided by Dr. Jeff Ranish (Institute for 
Systems Biology, Seattle, WA). 
 
 
 
The maintenance and expression of genetic information stored in DNA depends on the function of 
many essential enzymes and proteins.  The highly conserved general transcription factor TFIIH is 
required for transcription of DNA by RNA polymerase II (Pol II) as well as DNA repair.   TFIIH 
consists of ten protein subunits, several of which have enzymatic activity.  The XPB/Ssl2 and 
XPD/Rad3 (human/yeast) subunits are important for opening the DNA for transcription or repair, 
while the Cdk7/Kin28 kinase phosphorylates the C-terminal domain (CTD) of Pol II to initiate 
elongation.  
 
The structure and function of TFIIH complex components have been studied extensively 
biochemically; however, an overall map of its architecture has only recently been attempted. Dr. Jeff 
Ranish (Institute for Systems Biology), along with collaborators including Dr. Steve Hahn (Fred 
Hutch, Basic Sciences Division), knew that a high-resolution structure would identify specific protein-
protein interactions that are essential for TFIIH function.  
 
Thus, researchers in the Ranish Lab at ISB performed cross-linking/mass spectrometry (CXMS) on 
purified TFIIH complex from budding yeast and human cells, prepared in collaboration with 
researchers at University of Colorado, Boulder. In order to further increase their resolution, authors 
in the Sali Lab at UCSF used an integrative modeling approach to build a structure of 
TFIIH.  Integrative modeling finds solutions using all input data simultaneously and this often builds 
models that are more precise and complete.  Using this method, researchers in the Sali Lab used all 
the data available to them, including the CXMS data from the Ranish Lab, published EM densities, 
X-ray crystallographic data, as well as other types of data, to find solutions that fit well to a 
comprehensive model of human or yeast TFIIH.  Ultimately, they were able to identify the locations 
October 19, 2015 SCIENCE SPOTLIGHT 
 
2 Volume 5, Issue 10 | Fred Hutchinson Cancer Research Center 
 
and positions of each of the ten subunits of TFIIH, with the highest resolution for the protein 
XPD/Rad3 at 9.2Å and 17.7Å (human/yeast).  They observed that the overall organization of the 
subunits was very similar between yeast and human TFIIH, however there was a difference in the 
overall shape of the complex.  This could reflect differences in the preparation of the complexes for 
EM or may reflect real differences in structure.  
 
The authors were very interested in mapping conserved interfaces between subunits of TFIIH 
because combining the data could reveal essential interactions.  Interestingly, human disease 
mutations have been mapped to non-enzymatic components and therefore suggest that defects in 
the organization of the complex can have profound effects on the function of TFIIH.  The authors 
mapped their crosslinks onto the aligned sequences of human and yeast TFIIH and found that many 
represented novel interactions between specific domains within and between subunits.  Overall, the 
authors found four regional hubs of protein-protein interactions, naming them the Anchor, the Hub, 
the Lock and the Latch.  
 
The Anchor consists of an unstructured region of p62/Tfb1 that contacts domains in XPD/Rad3, 
p44/Ssl1, and p34/Tfb4.  The authors notice that there are not many crosslinks within p62/Tfb1 itself, 
suggesting that the unstructured Anchor region protrudes from the rest of protein.  The Hub region is 
found in the C-terminus of the p52/Tfb2 protein and connects the XPB/Ssl2 subunit to the base of 
TFIIH, the p44/Ssl1-p34/Tfb4 dimer.  The Lock region includes the N-terminal and C-terminal 
extensions of XPB/Ssl2 and shows a considerable number of interactions with other subunits as well 
as with each other.  The authors suggest that these interactions may regulate the enzymatic activity 
of XPB/Ssl2.  Finally, the authors found that the Latch region of MAT1/Tfb3 links Cdk7/Kin28-
CyclinH/Ccl1, a Cdk-Activating Kinase (CAK) module, to core TFIIH through interactions with 
XPB/Ssl2, XPD/Rad3 and p44/Ssl1.   
     
To further understand the function of the p62/Tfb1 protein, visiting Masters student Marine Dehecq 
(Université Paris) performed a deletion analysis of yeast Tfb1 while in the Hahn Lab (Basic Sciences 
Division) and found that a central part of the Anchor region is required for cell viability.  This is 
particularly striking given that deletion of other known interaction domains in Tfb1 is not lethal but 
instead causes sensitivity to UV light.  Furthermore, the authors showed that deletion of the Anchor 
domain in yeast Tfb1 and human p62 resulted in loss of co-IP with XPD/Rad3.  These results show 
that the Anchor region is essential for TFIIH structural integrity.   
 
Finally, the authors were able to map the position of disease-associated mutations in human patients 
with autosomal recessive diseases such as Xeroderma pigmentosum (XP) onto their model.  Their 
October 19, 2015 SCIENCE SPOTLIGHT 
 
3 Volume 5, Issue 10 | Fred Hutchinson Cancer Research Center 
 
structural data suggests that some mutations associated with XP specifically disrupt the interaction 
of the Anchor protein p62/Tfb1 with XPD/Rad3, and not p44/Ssl1 as previously suggested.  This 
work highlights the value of detailed structural information in the study of essential eukaryotic protein 
complexes.       
 
Luo J*, Cimermancic P*, Viswanath S, Ebmeier CC,  Kim B, Dehecq M, Raman V, Greenberg CH, 
Pellarin R, Sali A, Taatjes DJ, Hahn S, Ranish J.  2015.  Architecture of the human and yeast 
general transcription and DNA repair factor TFIIH.  Molecular Cell. 59:794-806.   
 
*co-first author 
 
This work was funded by the NIH, NSF, and HHMI.  
 
 
 


It takes ten to tango: the structure of transcription factor TFIIH.

Zentner, Gabriel E.

February 16, 2015 SCIENCE SPOTLIGHT 
 
1 Volume 5, Issue 2 | Fred Hutchinson Cancer Research Center 
 
No Origin, No Problem for Yeast DNA Replication 
February 16, 2015 
    GE Zentner 
To ensure proper transmission of genetic information, cells must accurately replicate their genome 
during each cell cycle.  In budding yeast, DNA replication initiates from well-defined origins called 
autonomously replicating sequences (ARSs), while in multicellular organisms replication it is thought 
to initiate from broader, less well-defined zones.  Interestingly, some recent work has suggested 
unexpected plasticity in the initiation of DNA replication, particularly in organisms with discrete 
origins of replication.  For instance, the archaeon Haloferax volcanii can replicate its chromosome 
even when its single origin is deleted, and origin-deficientHaloferax cells grow faster than wild-type 
cells (Hawkins et al., 2013).  Additionally, budding yeast lacking several ARSs on chromosome III 
show no appreciable increase in the loss of this chromosome during cell division (Dershowitz et al., 
2007).  Despite these observations, how DNA replication can initiate in the absence of ARSs is 
unclear.  To address this question, graduate student Naomi Bogenschutz and postdoctoral fellow Dr. 
Jairo Rodriguez in the lab of Dr. Toshio Tsukiyama (Basic Sciences Division) generated a yeast 
strain carrying a chromosome lacking seven well-characterized ARSs.  They found that this strain 
displayed no increase in sensitivity to DNA damaging agents and that DNA replication initiated from 
non-canonical sites on chromosome VI, suggesting that DNA replication in budding yeast is more 
flexible than previously thought. 
To study how yeast can replicate their DNA in the absence of ARSs, the authors generated a yeast 
strain in which seven ARSs on chromosome VI were deleted (hereafter referred to as 7oriΔ).  They 
first tested 7oriΔ for sensitivity to replication stress by growing it in medium containing stress-
inducing chemicals.  Strikingly, 7oriΔ cells showed no increase in sensitivity to replication stress, 
suggesting the presence of additional mechanisms for maintaining replication initiation in the 
absence of ARSs. 
Replication is initiated by the binding of the origin recognition complex (ORC) to origins, so the 
authors next investigated the binding of the ORC subunit Orc2 to chromosome VI in 
the 7oriΔ strain.  Consistent with the recognition of ARSs by ORC, Orc2 binding was strongly 
reduced at each origin deleted in 7oriΔ. However, low-level ORC binding adjacent to deleted origins 
was detected, indicated that ORC binding is not confined to ARSs. 
 
February 16, 2015 SCIENCE SPOTLIGHT 
 
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To investigate how replication could initiate in the absence of ARSs, the authors profiled replication 
using incorporation of bromodeoxyuridine (BrdU).  BrdU is an analog of the nucleotide thymidine that 
can be captured with an anti-BrdU antibody, allowing isolation of newly replicated DNA.  This 
revealed robust BrdU signal around each of the deleted origins, suggesting that residual levels of 
ORC bound adjacent to deleted origins might be sufficient for initiation.  Indeed, higher resolution 
analysis of initiation in 7oriΔ revealed that replication initiated from multiple sites adjacent to the 
deleted origins. 
These results reveal a new safeguard for replication integrity, wherein sequences adjacent to 
canonical origins are sufficient to initiate replication when the ARS is lost.  This may indicate that 
DNA replication origins in budding yeast are not as discrete as previously thought, and may be more 
similar to metazoan origins.  "Our results suggest that even in budding yeast, at least when a large 
number of origins fail, replication can initiate from non-canonical sites, which suggests that the 
difference between budding yeast and metazoan systems is not as black and white as people 
generally describe," said Dr. Tsukiyama.  "An interesting possibility is that we may be able to use the 
yeast mutant carrying this chromosome as a model for metazoan-like replication initiation." 
 
Bogenschutz NL, Rodriguez J, Tsukiyama T. 2014. Initiation of DNA Replication from Non-Canonical 
Sites on an Origin-Depleted Chromosome. PLOS ONE 9(12):e114545. 
 
See also: Hawkins M, Malla S, Blythe MJ, Nieduszynski CA, Allers T. 2013. Accelerated growth in 
the absence of DNA replication origins. Nature 503(7477):544-547. 
Dershowitz A, Snyder M, Sbia M, Skurnick JH, Ong LY, Newlon CS. 2007. Linear Derivatives of 
Saccharomyces cerevisiase Can Be Maintained in the Absence of Autonomously Replicating 
Sequence Elements. Mol Cell Biol 27(13):4652-4663. 
 
 
 
 
 
February 16, 2015 SCIENCE SPOTLIGHT 
 
3 Volume 5, Issue 2 | Fred Hutchinson Cancer Research Center 
 
 
 
 
Image from the publication 
(a) Schematic of chromosome VI, denoting the location of intact 
ARSs with grey arrows and deleted ARSs with black arrows. (b) 
Growth analysis of wild-type and 7oriΔ yeast on standard medium 
(top row) or medium containing various stressors (middle and 
bottom rows). The mec1Δ sml1Δ strain serves as a positive control 
for sensitivity to replication stress. 
 


No Origin, No Problem for Yeast DNA Replication

February 16, 2015 SCIENCE SPOTLIGHT 
 
1 Volume 5, Issue 2 | Fred Hutchinson Cancer Research Center 
 
The Curious Case of Helicobacter Pylori Csd4 
February 16, 2015 
     A Neves 
The bacterium Helicobacter pylori is estimated to persistently infect about half of the world’s 
population. While most cases are asymptomatic, H. pylori infection is a major risk factor for gastric 
ulcers and cancers. Previous work had identified genes that are important for the bacterium’s helical 
shape and efficient murine stomach colonization, and were collectively referred to as csd (cell shape 
determinant) genes (Sycuro et al., 2010, 2012). The csd genes encode proteins with a wide array of 
biochemical activities, highlighting the complexity of establishing and maintaining helical 
morphology. One of the csdgenes, csd4, was found to encode a carboxypeptidase that trims non-
cross-linked peptides in the peptidoglycan layer, a key structural scaffold in bacterial cells. Deletion 
of csd4 resulted in straight or slightly curved rods with impaired stomach colonization in murine 
models of H. pylori infection (Sycuro et al., 2012). However, it has remained unclear how Csd4 
regulates helical shape. A new Fred Hutch study led by graduate student Kris Blair in the lab of Dr. 
Nina Salama (Human Biology Division) and collaborators from the University of British Columbia, 
solved the structure of Csd4 in order to elucidate its mechanism of action. Their results were recently 
published in the Journal of Biological Chemistry. 
The 1.40 Å resolution crystal structure revealed an N-terminal carboxypeptidase domain (pink in 
panel B of figure), followed by two smaller domains of unknown function that were found to be 
required for stable Csd4 expression (blue and yellow in panel B of figure). Since the initial crystal 
structure did not contain bound zinc in the active site, the authors soaked Csd4 crystals in zinc, 
which resulted in changes in active site ligands. Another crystal solved to 1.75 Å resolution in the 
presence of a tripeptide substrate, maintained the overall fold of the zinc-only crystal. Next, the 
investigators examined the role of glutamine 46 (Q46) in Csd4’s catalytic activity by constructing 
three active site variants (Q46H, Q46A and Q46E) and found that all three variants had impaired 
enzymatic activity, though the Q46H mutant retained about 50% activity in some buffer conditions. 
To probe the role of Q46 in normal helical shape, the authors generated H. pylori strains expressing 
either Q46H or Q46A at the native locus and discovered that neither one of the mutant proteins were 
able to rescue the helical shape defect of a csd4 loss-of-function mutant, even when present in two 
copies. Finally, the researchers created a strain expressing both a wild-type csd4 and 
a csd4Q46H allele and observed a dominant-negative effect, meaning that the CsdQ46H protein 
prevented the wild-type Csd4 from performing its normal function. Intriguingly, the Q46H allele still  
February 16, 2015 SCIENCE SPOTLIGHT 
 
2 Volume 5, Issue 2 | Fred Hutchinson Cancer Research Center 
 
 
increased cell length, suggesting that not all of Csd4’s functions require its enzymatic activity. 
"From our perspective this paper is interesting for a number of reasons. First, the crystal structure 
revealed two C-terminal domains of unknown function that we found to be required for stable Csd4 
expression. Second, and as the title of the paper implies, the active site of H. pylori Csd4 contains 
an atypical glutamine in place of a generally conserved histidine found in related carboxypeptidases. 
Much to our surprise, substitution with the histidine was insufficient to restore protein function in vivo. 
Overexpression of the Csd4-Q46H variant was unable to rescue helical cell shape but led to an 
increase in cell-body axis length, indicating a possible role for domains 2 and 3 in cell elongation. 
Co-expression of both variants in the same cell resulted in non-helical bacteria suggesting 
cooperativity of Csd4 protein as part of a protein complex. Curiously, this conserved site seems to 
be toggling back and forth between the two residues in the Epsilonproteobacteria, but does not 
appear to track with the cell shape of the organism in which it is found," summarized Mr. Blair. 
Overall, this study presented mechanistic insight into how Csd4 functions by solving its substrate-
bound crystal structure and by demonstrating that glutamine, an uncommon zinc ligand, is essential 
for enzymatic activity and for the normal helical shape of H. pylori. It also suggests that Csd4 is a 
founding member of a new family of carboxypeptidades. 
 
Chan ACK, Blair KM, Liu Y, Frirdich E, Gaynor EC, Tanner ME, Salama NR, Murphy MEP. 2014. 
Helical Shape of Helicobacter pylori Requires an Atypical Glutamine as a Zinc Ligand in the 
Carboxypeptidase Csd4. J Biol Chem. 290(6):3622-38. 
Sycuro LK, Wyckoff TJ, Biboy J, Born P, Pincus Z, Vollmer W, Salama NR. 2012. Multiple 
peptidoglycan modification networks modulate Helicobacter pylori's cell shape, motility, and 
colonization potential. PLoS Pathog 8(3):e1002603. 
Sycuro LK, Pincus Z, Gutierrez KD, Biboy J, Stern CA, Vollmer W, Salama NR.2010. Peptidoglycan 
crosslinking relaxation promotes Helicobacter pylori's helical shape and stomach 
colonization. Cell 141(5):822-33. 
 
 
 
February 16, 2015 SCIENCE SPOTLIGHT 
 
3 Volume 5, Issue 2 | Fred Hutchinson Cancer Research Center 
 
 
 
 
Image provided by Mr. Blair, Ms. Jenny Taylor and Scientific 
Imaging 
A. Transmission electron micrograph of wild-type Helicobacter 
pylori. B. Crystal structure of Csd4 showing three-domain 
architecture (catalytic domain is pink and c-terminal domains of 
unknown function are blue and yellow). C. Scatter plot of cell 
side-curvature vs. cell axis-length of wild-type and mutant h. 
pylori bacteria (>200 cells/plot). Strain labels indicate the copy 
number (1 or 2) and amino acid residue at position 46 (1 or 2). 
 


The Curious Case of Helicobacter Pylori Csd4

Pattacini, Laura

March 16, 2015 SCIENCE SPOTLIGHT 
 
1 Volume 5, Issue 3 | Fred Hutchinson Cancer Research Center 
 
Shadows Looming Over Tenofovir Gel Application 
March 16, 2015 
    L Pattacini 
The efficacy of the antiretroviral drug tenofovir, a phosphonate nucleoside analogue HIV reverse 
transcriptase inhibitor (NRTI), for HIV prevention in situations of high exposure has been tested in a 
number of trials. While the oral pill, alone or in combination with emtricitabine (Truvada®), has 
proven to provide protection, studies testing tenofovir gel applications have yielded mixed results. 
Lack of protection was mostly attributed to low adherence, but off-target effects of the drug on the 
mucosa could have contributed. Moreover, during topical use of antiretrovirals, very high local drug 
concentrations are achieved, warranting a comprehensive assessment of the mucosal changes 
induced by each candidate microbicide to ensure safety.  
To address this question, Dr. Florian Hladik from the Vaccine and Infectious Disease Division 
(VIDD), in a joint effort with other investigators from Fred Hutch as well as other institutions in US 
and in Canada, conducted a systems biology study within the clinical trial MTN-007 (Protocol Chair: 
Dr. Ian McGowan, University of Pittsburgh), in which 65 participants were randomized to receive a 
rectal gel formulation containing tenofovir 1%, nonoxynol-9 (N-9) 2% (a detergent included as a 
positive toxicity control), no gel or no treatment. Furthermore, as tenofovir gel is a candidate product 
for HIV prevention in women, its effect on vaginal keratinocytes obtained from tissues discarded 
following vaginal repair surgery was also evaluated. "Topical microbicides, for example vaginal gels 
containing an antiviral drug, are an entirely new HIV prevention strategy for which no precedence 
exists.  So it is prudent to broadly assess their safety, in particular given the high drug 
concentrations that are sometimes achieved in the mucosa" explained Dr. Hladik. The clinical study 
was reported in PLOS One in 2013; the results of the systems biology study and follow-up 
experiments were published in the February edition of eLIFE. 
The effect of tenofovir gel on global gene expression profile was determined in rectal biopsies as 
well as in primary vaginal keratinocyte cultures. In the rectal mucosa, one week of daily tenofovir 
applications suppressed the expression of 505 genes and induced 137 genes. The effect of N-9 was 
much more limited in that it suppressed only 56 and induced 60 genes. Among the genes highly 
activated by tenofovir gel, the investigators identified several encoding for secretory proteins, such 
as the chemokines CCL2, CCL19, CCL21, CCL23, CXCL9 and CXCL13; correspondingly, a number 
of lymphocyte markers were increased. On the other hand, tenofovir inhibited the transcription of 
many genes encoding for transcription factors, as well as genes regulating cell proliferation,  
March 16, 2015 SCIENCE SPOTLIGHT 
 
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apoptosis and epithelial structure organization. Tenofovir also suppressed immunoregulatory 
molecules such as interleukin 10 (IL-10), a cytokine important for toning down inflammatory 
processes (see Figure).  A similar pattern of gene expression modification was detected in cultures 
of vaginal keratinocytes treated with tenofovir in vitro. These cells also grew faster under the 
influence of tenofovir, indicating a hyperplastic effect of the drug.  
Both in vivo and in vitro, tenofovir also suppressed the expression of PNPT1, a master regulator of 
RNA import into mitochondria. This observation led the investigators to explore the possibility that 
tenofovir affects mitochondrial functions. They observed a decrease in the transcription of 
mitochondrial DNA, and in two samples analyzed by electron microscopy found that the number of 
mitochondria significantly decreased. Mitochondrial toxicity of NRTIs is well known but as yet 
unexplained – the current data point to blockade of PNPT1 as a possible cause.   
In conclusion, this study uncovered a number of potentially concerning effects of the antiretroviral 
drug tenofovir on the mucosa, although concrete clinical correlates of these activities are still lacking. 
Should tenofovir 1% gel become available as an HIV prevention tool, its safety over long-term use 
must be carefully monitored. The immunological, mitochondrial and hyperplastic properties of 
tenofovir at high concentrations could limit its usefulness as a topical microbicide and argue in favor 
of alternative drugs with a different mechanism of action. 
"Our study shows that a broad systems biology approach can uncover potential safety concerns that 
need to be followed up upon.  We also gained a lot of new information about the biology of the 
human vaginal and rectal mucosa, which will keep us busy for many years to come" concluded Dr. 
Hladik. His Lab is already planning follow-up studies, and we look forward to reporting future exciting 
results from this work. 
 
Hladik F, Burgener A, Ballweber L, Gottardo R, Vojtech L, Fourati S, Dai JY, Cameron MJ, Strobl J, 
Hughes SM, Hoesley C, Andrew P, Johnson S, Piper J, Friend DR, Ball TB, Cranston RD, Mayer 
KH, McElrath MJ, McGowan I. 2015. Mucosal effects of tenofovir 1% gel. Elife, 4. doi: 
10.7554/eLife.04525. 
 
 
 
March 16, 2015 SCIENCE SPOTLIGHT 
 
3 Volume 5, Issue 3 | Fred Hutchinson Cancer Research Center 
 
 
 
 
 
Image provided by Dr. Florian Hladik. 
Immunohistochemistry visualization of IL10 production at day O and 
7 of Tenofovir treatment in colon mucosa. 
 


Shadows Looming Over Tenofovir Gel Application

Kocarnik, Jonathan

April 20, 2015 SCIENCE SPOTLIGHT 
 
1 Volume 5, Issue 4 | Fred Hutchinson Cancer Research Center 
 
Common Supplements Found To Lower 
Circulating Inflammation Levels 
April 20, 2015 
    JM Kocarnik 
Glucosamine and chondroitin are among the most commonly used non-vitamin dietary supplements 
in the United States.  Typically used for joint pain, these supplements are often taken together as a 
single pill.  Previous studies have suggested that may play a role in lowering inflammation, which 
may in turn lower risk for lung and colorectal cancer.  In order to evaluate the effect of these 
supplements on systemic inflammation levels, Drs. Sandi Navarro, Johanna Lampe, Emily White, 
and colleagues in the Public Health Sciences Division conducted a randomized crossover trial.  As 
recently reported in PLoS One, the researchers found that glucosamine and chondroitin 
supplementation lowered C-reactive protein concentrations by 23% compared to placebo. 
Chronic inflammation contributes to several diseases, including several types of cancer.  One of the 
major components of the inflammation process is nuclear factor kappa B (NF-κB), a transcription 
factor important for the generation of cytokines, chemokines, and other factors involved in the 
immune response.  Previous studies have suggested that glucosamine and chondroitin may help 
reduce inflammation by inhibiting NF-κB and reducing oxidative stress.  To further investigate this 
potential relationship, the authors measured the effect of glucosamine and chondroitin 
supplementation on several inflammatory biomarkers.  These included C-reactive protein (CRP), 
interleukin-6 (IL-6), the soluble tumor necrosis factor receptors I and II (sTNFRI and sTNFRII), and 
two urinary metabolites.  
A crossover trial design was used, where each participant was randomized to receive either the 
supplements or the placebo, followed by a washout period of taking nothing, and then switched to 
the other pill.   Measurements for inflammatory biomarkers were taken at each stage. "We found that 
glucosamine and chondroitin lowered C-reactive protein by 23% compared to an inactive pill," said 
lead author Dr. Sandi Navarro.  None of the other biomarkers showed a significant difference.  In 
addition to measuring inflammatory biomarker concentrations, the researchers also evaluated 
changes in various plasma proteins by using a 3000-antibody microarray.  "When we looked at a 
broad range of proteins in the blood together, we found that 'cytokine activity' was the pathway with 
the largest difference between the glucosamine and chondroitin intervention and the inactive pill, and 
was lower after glucosamine and chondroitin supplementation."  Alterations seen in additional  
April 20, 2015 SCIENCE SPOTLIGHT 
 
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inflammation-related pathways further suggested that these supplements may have wider-ranging 
effects on inflammation than just NF-κB inhibition. 
Together, these results agree with previous evidence suggesting that these supplements have anti-
inflammatory properties that may lower cancer risk.  "This is the first randomized trial to evaluate the 
effects of glucosamine and chondroitin on inflammation in healthy adults and the first to assess 
potential pathways that may be affected by using a broad protein screen and pathway analyses," 
said Dr. Navarro.  "The trial was motivated by results in the VITAL study—a large prospective cohort 
of 77,738 adults specifically designed to evaluate the association of supplement use and cancer. Of 
the 38 supplements studied, the most consistent results in terms of  cancer risk reduction were for 
use of glucosamine and chondroitin,  including a 26% reduction in lung cancer risk, a 27% reduced 
risk of colorectal cancer, and a 13% reduced risk in cancer mortality." 
Future, larger studies will be needed to further support these findings, as well as to evaluate whether 
these anti-inflammatory effects are seen in a broader range of individuals.  In the meantime, this 
study suggests that these supplements could potentially be useful for more than joint pain.  Said Dr. 
Navarro, "these results may provide a possible biologic mechanism to support our previous findings 
that use of glucosamine and chondroitin is associated with reduced lung and colon cancer and 
overall mortality." 
Other researchers from the Public Health Sciences Division contributing to this project were Drs. 
Junghyun Rho, Xiaoling Song, and Paul Lampe, as well as Ms. Yuzheng Zhang. 
 
Citation:  
Navarro SL, White E, Kantor ED, Zhang Y, Rho J, Song X, Milne GL, Lampe PD, Lampe JW. 2015. 
Randomized trial of glucosamine and chondroitin supplementation on inflammation and oxidative 
stress biomarkers and plasma proteomics profiles in healthy humans. PLoS One. 10(2):e0117534.  
 
 
 
 
 
April 20, 2015 SCIENCE SPOTLIGHT 
 
3 Volume 5, Issue 4 | Fred Hutchinson Cancer Research Center 
 
 
 
 
Image provided by Dr. Sandi Navarro 
Hypothesized mechanism of glucosamine and chondroitin on 
inflammation and oxidative stress. 
 


Common Supplements Found To Lower Circulating Inflammation Levels

Zentner, Gabriel

April 20, 2015 SCIENCE SPOTLIGHT 
 
1 Volume 5, Issue 4 | Fred Hutchinson Cancer Research Center 
 
Wash: Not Just For the Cytoplasm Anymore 
April 20, 2015 
    GE Zentner 
Organization of the cytoskeleton is critical for maintenance of cell shape and wound repair, among 
other processes.  Key regulators of the cytoskeleton include Wiskott-Aldrich syndrome family (WAS) 
proteins, which regulate actin polymerization and are often misregulated in immune disorders and 
metastasis (Rottner et al., 2010).  While studying the cytoplasmic functions of the Drosophila WAS 
protein Washout (Wash), research associate Jeffrey Verboon and colleagues in the laboratory of Dr. 
Susan Parkhurst (Basic Sciences Division) made the surprising finding that a substantial amount of 
Wash is present in the nucleus.  "We found an unexpected interaction [of Wash] with B-type Lamin, 
which led us to collaborate with the Groudine lab, who have expertise in all things nuclear.  Together 
with co-first-author Dr. Hector Rincon, we characterized Wash's nuclear defects finding global 
changes to the shape of the nucleus, components which mark nuclear compartments, PEV and 
chromatin accessibility," said Mr. Verboon. 
Following up on their initial observation of significant localization of Wash to the nucleus, the authors 
found that Wash associates with several hundred sites on Drosophilapolytene chromosomes, 
despite lacking a DNA binding domain.  To address potential functions of nuclear Wash, the 
researchers knocked it down using RNA interference (RNAi) in cell culture.  Wash depletion led to 
abnormal nuclear morphology, and this observation was confirmed in salivary glands. 
The authors next asked if loss of Wash might affect nuclear organization.  Microscopic examination 
of polytene chromosomes from wash mutants revealed a general lack of organization as well as 
increased susceptibility to breakage.  Mutant chromosomes also tended to cluster at the nuclear 
periphery, in contrast to the well-defined chromosome territories observed in wild-type cells.  It was 
also found that HP1, which marks heterochromatin, only weakly accumulated on chromosomes 
in wash mutants.  The organization of the nucleolus and Cajal bodies, both nuclear organelles, was 
dispersed in wash mutants.  Lastly, the authors found that histone modifications associated with both 
activation and repression of transcription were diminished in wash mutants.  The requirement for 
nuclear localization of Wash in nuclear organization was confirmed by the observation of aberrant 
nuclear morphology in cells expressing a version of Wash lacking its consensus nuclear localization 
signal. 
The authors identified a strong interaction between Wash and Lamin, a filament protein that forms a 
mesh that lines the inner nuclear membrane and has key functions in maintaining nuclear shape and  
April 20, 2015 SCIENCE SPOTLIGHT 
 
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regulating nuclear organization and gene expression.  Using the DamID method to identify Wash 
binding sites across the genome, the authors found a strong concordance between Wash-
associated regions and Lamin-associated domains (LADs), suggesting a functional interaction 
between the two proteins on chromatin, particularly at regions of heterochromatin, which are often 
within LADs. 
Given that wash mutants displayed general chromosomal disorganization and reductions in histone 
marks, the authors hypothesized that Wash impacts chromatin structure.  Indeed, Wash knockdown 
in cell culture resulted in increased accessibility at the boundaries of heterochromatic regions.  The 
increase in heterochromatin accessibility observed with Wash depletion was confirmed in vivo using 
position-effect variegation (PEV) reporter, which assays the ability of a protein to influence chromatin 
in different contexts.  Consistent with expectations, wash mutants enhanced centromeric and 
heterochromatic PEV. 
"What's really interesting, and the next step, is the mechanism by which these nuclear phenotypes 
are caused.  Perhaps, the global defects are the consequence of a single process, for instance if 
Wash is a key player in the nucleoskeleton or in trafficking things inside of the nucleus, analogous to 
its roles with the cytoskeleton and endocytic trafficking in the cytoplasm.  On the other hand, Wash 
may have many roles each leading to specific defects which overall read out as a global regulator," 
said Mr. Verboon.  "Our next steps will be to use proteomics in tandem with cell and molecular 
approaches to tease out the mechanism(s) for the striking phenotypes we see." 
 
Verboon JM, Rincon-Arano H, Werwie TR, Delrow JJ, Scalzo D, Nandakumar V, Groudine M, 
Parkhurst SM. 2015. Wash interacts with Lamin and affects global nuclear organization. Curr 
Biol 25(6):804-810. 
See also: Rottner K, Hänisch J, Campellone KG. 2010. WASH, WHAMM and JMY: regulation of 
Arp2/3 complex and beyond. Trends Cell Biol 20(11):650-651. 
 
 
 
 
April 20, 2015 SCIENCE SPOTLIGHT 
 
3 Volume 5, Issue 4 | Fred Hutchinson Cancer Research Center 
 
 
 
Image from the publication 
(Top left) A cell from a wild-type salivary gland stained for Lamin, 
demonstrating normal nuclear architecture. (Bottom left) A cell from 
a wash mutant salivary gland stained for Lamin, demonstrating 
abnormal nuclear organization. (Top right) A cell from a salivary 
gland expressing wild-type Wash and stained for Lamin (green) and 
coilin, a marker for Cajal bodies (red), demonstrating normal Cajal 
body morphology. (Bottom right) A cell from a salivary gland 
expressing nuclear localization-deficient Wash and stained for 
Lamin (green) and coilin (red), demonstrating aberrant Cajal body 
morphology. 
 


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