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Novel recognition site for polyUb and UBL signals in Rpn1

ABSTRACT: Rpn1 is the largest proteasomal receptor for polyubiquitin and shuttle proteins contains one known binding site for polyubiquitin and shuttle proteins. We used a combination of NMR spectroscopy, photo-crosslinking, mass spectrometry, and mutagenesis,to confirm the presence of additional ubiquitin recognition site on RPN1. This novel site is situated in the N-terminal section of Rpn1, a region previously surmised to be devoid of functionality.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human) Saccharomyces Cerevisiae (baker's Yeast)

SUBMITTER: Oded Kleifeld

LAB HEAD: David Fushman

PROVIDER: PXD027128 | Pride | 2021-08-09

REPOSITORIES: Pride

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Promoters Active in Interphase are Bookmarked during Mitosis by Ubiquitination

Project description:We analyzed genome-wide modification of chromatin by ubiquitination in human cells and whether this mark changes through the cell cycle using modified ChIP-seq technique (ChAP-seq). Chromatin affinity purification was based on a standard ChIP method with modification of a two-step affinity purification.

2013-04-04 | E-GEOD-45771 | biostudies-arrayexpress

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Project description:This SuperSeries is composed of the SubSeries listed below. Refer to individual Series

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HDACs act on ribosomal DNA to control the yeast replication program and the competition between origins for limiting initiation factors [ChIP-seq]

Project description:In S. cerevisiae, replication timing is controlled by epigenetic mechanisms restricting the accessibility of origins to limiting initiation factors. About 30% of these origins are located within repetitive DNA sequences such as the ribosomal DNA (rDNA) array, but their regulation is poorly understood. Here, we have investigated how histone deacetylases (HDACs) control the replication program in budding yeast. This analysis revealed that two HDACs, Rpd3 and Sir2, control replication timing in an opposite manner. Whereas Rpd3 delays initiation at late origins, Sir2 is required for the timely activation of early origins. Moreover, Sir2 represses initiation at rDNA origins whereas Rpd3 counteracts this effect. Remarkably, deletion of SIR2 restored normal replication in rpd3 cells by reactivating rDNA origins. Together, these data indicate that HDACs control the replication timing program in budding yeast by modulating the ability of repeated origins to compete with single-copy origins for limiting initiation factors. BrdU-IP-seq analysis of origin activity in wt, sir2 and rpd3 cells, aligned against genomic DNA (sacCer3) and rDNA sequences Please note that the wt WCE #1 and #2 samples are whole-cell extracts from wild-type cells arrested in HU that were used to calculate the log ratio of the BrdU IP #1 and #2 batches, respectively.

2014-05-14 | E-GEOD-57617 | biostudies-arrayexpress

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Project description:Comparative hydrogen deuterium exchange mass spectrometry of WT and I507A mutant for above titled manuscript.

2022-02-17 | PXD030382 | Pride

HDACs act on ribosomal DNA to control the yeast replication program and the competition between origins for limiting initiation factors

Project description:In S. cerevisiae, replication timing is controlled by epigenetic mechanisms restricting the accessibility of origins to limiting initiation factors. About 30% of these origins are located within repetitive DNA sequences such as the ribosomal DNA (rDNA) array, but their regulation is poorly understood. Here, we have investigated how histone deacetylases (HDACs) control the replication program in budding yeast. This analysis revealed that two HDACs, Rpd3 and Sir2, control replication timing in an opposite manner. Whereas Rpd3 delays initiation at late origins, Sir2 is required for the timely activation of early origins. Moreover, Sir2 represses initiation at rDNA origins whereas Rpd3 counteracts this effect. Remarkably, deletion of SIR2 restored normal replication in rpd3 cells by reactivating rDNA origins. Together, these data indicate that HDACs control the replication timing program in budding yeast by modulating the ability of repeated origins to compete with single-copy origins for limiting initiation factors. BrdU-IP-chip analysis of origin usage in different yeast HDAC mutants

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2022-01-19 | PXD027639 | Pride

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CTCF loss has limited effects on genome architecture in Drosophila despite critical regulatory functions.

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2023-03-11 | PXD032977 | Pride

Integrated approaches reveal determinants of genome-wide binding and function of the transcription factor Pho4

Project description:Binding of transcription factors to DNA is a key regulatory step in the control of gene expression. DNA sequences with high affinity for transcription factors occur more frequently in the genome than instances of genes bound or regulated by these factors. Although several mechanisms have been identified that influence the specificity of transcriptional regulation, it is not known if these can explain the observed genome-wide pattern of binding or regulation for a given transcription factor. We used genome-wide approaches to study how trans influences shape the binding and regulatory landscape of Pho4, a budding yeast transcription factor that activates gene expression in response to phosphate limitation. We find that nucleosomes significantly restrict the sites to which Pho4 binds. At nucleosome-depleted sites, competition between Pho4 and another transcription factor, Cbf1, determines Pho4 occupancy, raising the threshold for transcriptional activation by Pho4 in phosphate replete conditions and preventing Pho4 activation of genes outside the phosphate regulon during phosphate starvation. Pho4 binding is not sufficient for transcriptional activation - a cooperative interaction between the transcription factor Pho2 and Pho4 occurs specifically at genes that are activated. Combining these experimental observations, we are able to globally predict Pho4 binding and its functionality. Our study provides insights into the mechanisms of global control by sequence-specific transcription factors. ChIP-Seq experiments of Pho4, Pho2 and Cbf1 samples and paired-end nucleosome sequencing in no Pi conditions.

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