1,721,035 research outputs found
Single nucleosome analysis of human cell-cycle CCAAT promoters in vivo : a positive role for KDM1
No full textThe CCAAT box is a frequent promoter element, as illustrated by bioinformatic analysis of large sets of promoters, and it is bound by NF-Y which is involved in positive, as well as negative regulation. NF-Y is responsible of the transcriptional regulation of cell-cycle genes: it is an ubiquitous histone-like trimer composed of three subunits, NF-YA, NF-YB, and NF-YC, all necessary for DNA binding. NF-YB and NF-YC contain histone fold motifs (HFM), found in all core histones, whose dimerization is a prerequisite for NF-YA association, that confers to the trimer sequence-specific DNA binding capacity.
NF-Y binding is a prerequisite for promoter activation and allows further activators/co-activators buildup.
Here, we used a MNase I-based ChIP protocol on homogeneous cell populations to study histone modifications on cell-cycle promoters at a single nucleosome level in vivo.
Core regions of active promoters have H3-H4 and are depleted of H2A-H2B, substituted by NF-Y. A complex profile of H3K4 methylations is observed: H3K4me3 increases in active conditions, but significant levels are present on inactive promoters; H3K4me2 is relatively constant. Surprisingly, H3K4me1 best correlates with transcription. Analysis of histone demethylases suggests a positive role of KDM1(LSD1) and KDM5A in transcription, whereas KDM5B is present in repressed states. The use of the KDM1 inhibitor Tranylcipromine confirms that KDM1 is indeed involved in activation of G2/M promoters.
NF-Y removal by adenovirus infection has dramatic negative effects on H3K4me3, H3K79me2, H3K36me3, H4K79me3 and leads to the appearance of H4K20me3 and recruitment of KDMs. NF-Y recruits the KDM1 partner CoREST through direct interaction. These data are the first indication that KDM1 partakes in activation of cell-cycle genes and place NF-Y at the heart of regulation of key H3K4 methylations
Chromatin configuration of CCAAT-containing cell cycle promoters
No full textThe CCAAT box is a frequent promoter element bound by NF-Y, a trimer with H2A-H2B-like subunits. We developed a MNase I-based ChIP protocol on homogeneous cell populations to study cell-cycle promoters at the single nucleosome level. We analyzed histone acetylations and methylations and the association of enzymatic activities. This thesis presents different novel findings. The first finding was that H3-H4 take part of core promoters under active conditions, with the expected cohort of “positive” modifications, while H2A-H2B are removed and substituted by NF-Y. Through the use of a dominant negative mutant we show also that NF-Y is important for H3K36me3 deposition and for Pol II elongation. The second finding was that H3K4 methylations are highly dynamic and H3K4me1 is a crucial positive mark. Both functional and pharmacological inactivation led to state that KDM1 plays a positive role in transcription of G2/M genes. It requires CoREST, which is recruited on active promoters through direct interactions with NF-Y. Therefore, these preliminary data are the first in vivo indication of a crucial interplay between core histones and “deviant” histone-fold such as NF-Y, leading to fine tuning of histone methylations. The third finding was that NF-Y is not involved in histone acetyl-marks deposition as well as in histone methyl-marks: in fact, histone acetylation status of active cell cycle genes was only slightly perturbed after NF-Y removal. Moreover, this work proposed a special histone acetylation pattern typical of cell cycle gene cluster, characterized by H2BK120ac and H3K9,18,36ac deposited on H3 in repressive conditions. And finally, by in vivo analysis we showed GCN5 and PCAF involvement in the acetyl-marks deposition, and the probable NF-Y dependent recruitment of multisubunit complexes responsible of the chromatin remodelling, like STAGA and ATAC
NF-Y substitutes H2A-H2B on active cell-cycle promoters: recruitment of CoREST-KDM1 and fine-tuning of H3 methylations
No full textThe CCAAT box is a frequent promoter element, as
illustrated by bioinformatic analysis, and it is bound
by NF-Y, a trimer with H2A-H2B-like subunits.
We developed a MNase I-based ChIP protocol on
homogeneous cell populations to study cell-cycle
promoters at the single nucleosome level. We analyzed
histone methylations and the association of
enzymatic activities. Two novel results emerged:
(i) H3-H4 are present on core promoters under
active conditions, with the expected cohort of ‘positive’
modifications; H2A-H2B are removed and substituted
by NF-Y. Through the use of a dominant
negative mutant we show that NF-Y is important
for H3K36me3 deposition and for elongation, not
recruitment of Pol II; (ii) H3K4 methylations are
highly dynamic and H3K4me1 is a crucial positive
mark. Functional siRNA inactivation and treatment
with Tranylcypromine determined that KDM1 (LSD1)
plays a positive role in transcription, specifically of
G2/M genes. It requires CoREST, which is recruited
on active promoters through direct interactions with
NF-Y. These data are the first in vivo indication
of a crucial interplay between core histones and
‘deviant’ histone-fold such as NF-Y, leading to
fine-tuning of histone methylations
NF-Y-dependent regulation of chromatin structure on cell cycle promoters
No full textNF-Y is an ubiquitous trascription factor comprising three subunits, NF-YA, NF-YB, and NF-YC. NF-YB and NF-YC, H2A/H2B like, contain histone fold motifs (HFM), found in all core histones, whose dimerization is a prerequisite for NF-YA association, that confers to the trimer CCAAT-specific DNA binding capacity.
NF-Y binding is a prerequisite for promoter activation, as it pre-sets the promoter architecture in the near proximity of transcriptional start site allowing other regulatory proteins to access it. ChIP experiments determined that NF-Y binding to cell-cycle genes occurs in vivo before gene activation and in a very strictly regulated manner.
To investigate the role of NF-Y in the regulation of chromatin structure on cell cycle promoters, we performed ChIP analysis on cycling, G2/M blocked and G1 synchronized HCT116 cells, by using chromatin micrococcal digested.
From a fine nucleosome mapping of human CyclinB2 (marker of G2/M phase) and PCNA (G1 phase) we found a complementary pattern of expression between NF-Y subunits and core histones: far from the trascriptional start site enrichment is greater in core histones than in NF-Y subunits, while getting closer to the trascriptional start site the situation is reversed. Predictably, this is observed in G2/M blocked cells on CyclinB2 promoter, whereas on the PCNA promoter occurs in G1 synchronized cells. At the opposite, we observed H2A/H2B on CyclinB2 core promoter in G1 cells and on PCNA in G2/M cells. H3/H4 behaves differently from H2A/H2B, as they are enriched either on the core promoter or far from it.
We conclude that NF-Y acts not only as a classical trascription factor, but it also has a crucial role in nucleosomes remodelling within active chromatin
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
NF-Y affects histone acetylation and H2A.Z deposition in cell cycle promoters
No full textHistones post-translational modifications (PTMs) are crucial for transcriptional control, defining positive and negative chromatin territories. We previously described an extensive methylation-acetylation switch on cell cycle promoters using a single nucleosome ChIP assay. A key issue is how PTMs are locally positioned. We report an analysis on the role of the NF-Y CCAAT transcription factor on histone acetylation. Whereas H3K9 and H3K14 acetylation in core promoters is not influenced by NF-Y, H3K18ac, H3K36ac and H3K27ac are increased in the absence of NF-Y. Interestingly, NF-Y affects H2B acetylation in an opposite way: H2BK16ac is decreased and Lysine 120 acetylation, which counter-correlates with ubiquitination, increases dramatically upon NF-Y removal. KAT2A/KAT2B and subunits of the SAGA and ATAC complexes (SPT20 and ZZZ3) are differentially regulated. Finally, the deposition of H2A.Z, which maps around the TSS, is also NF-Y-dependent. In summary, NF-Y influences histone acetylation in different processes, including those involved in a methylation-acetylation switch and in the recruitment of histone variants
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