Chromatin Preparation and Chromatin Immuno-precipitation from Drosophila Embryos.

This protocol provides specific details on how to perform Chromatin immunoprecipitation (ChIP) from Drosophila embryos. ChIP allows the matching of proteins or histone modifications to specific genomic regions. Formaldehyde-cross-linked chromatin is isolated and antibodies against the target of interest are used to determine whether the target is associated with a specific DNA sequence. This can be performed in spatial and temporal manner and it can provide information about the genome-wide localization of a given protein or histone modification if coupled with deep sequencing technology (ChIP-Seq).

Proteomic data on the nuclear interactome of human MCM9.

We present data relating to the interactome of MCM9 from the nuclei of human cells. MCM9 belongs to the AAA+ superfamily, and contains an MCM domain and motifs that may confer DNA helicase activity. MCM9 has been shown to bind MCM8, and has been implicated in DNA replication and homologous recombination. However, the mechanistic basis of MCM9's role in DNA repair is poorly understood, and proteins with which it interacts were hitherto unknown. We performed tandem affinity purification of MCM9 and its interacting proteins from nuclear extracts of human cells, followed by proteomic analysis, thereby generating a set of mass spectrometry data corresponding to the MCM9 interactome [1]. The proteomic data set comprises 29 mass spectrometry RAW files, deposited to the ProteomeXchange Consortium, and freely available from the PRIDE partner repository with the data set identifier PXD000212. A set of 22 interacting proteins identified from the proteomic data was used to create an MCM9-centered interactive network diagram, using the Cytoscape program. These data allow the scientific community to access, mine and explore the human nuclear MCM9 interactome.

MCM9 Is Required for Mammalian DNA Mismatch Repair.

DNA mismatch repair (MMR) is an evolutionarily conserved process that corrects DNA polymerase errors during replication to maintain genomic integrity. In E. coli, the DNA helicase UvrD is implicated in MMR, yet an analogous helicase activity has not been identified in eukaryotes. Here, we show that mammalian MCM9, a protein involved in replication and homologous recombination, forms a complex with MMR initiation proteins (MSH2, MSH3, MLH1, PMS1, and the clamp loader RFC) and is essential for MMR. Mcm9-/- cells display microsatellite instability and MMR deficiency. The MCM9 complex has a helicase activity that is required for efficient MMR since wild-type but not helicase-dead MCM9 restores MMR activity in Mcm9-/- cells. Moreover, MCM9 loading onto chromatin is MSH2-dependent, and in turn MCM9 stimulates the recruitment of MLH1 to chromatin. Our results reveal a role for MCM9 and its helicase activity in mammalian MMR.

Identification of histone 3 variant 2 interacting factors.

The epigenome is defined as a type of information that can be transmitted independently of the DNA sequence, at the chromatin level, through post-translational modifications present on histone tails. Recent advances in the identification of histone 3 variants suggest a new model of information transmission through deposition of specific histone variants. To date, several non-centromeric histone 3 variants have been identified in mammals. Despite protein sequence similarity, specific deposition complexes have been characterized for both histone 3.1 (H3.1) and histone 3.3 (H3.3), whereas no deposition complex for histone 3.2 (H3.2) has been identified to date. Here, we identified human H3.2 partners by immunopurification of nuclear H3.2 complexes followed by mass spectrometry analysis. Further biochemical analyses highlighted two major complexes associated with H3.2, one containing chromatin associated factor-1 subunits and the other consisting of a subcomplex of mini chromosome maintenance helicases, together with Asf1. The purified complexes could associate with a DNA template in vitro.

Spt6 levels are modulated by PAAF1 and proteasome to regulate the HIV-1 LTR.

BACKGROUND: Tat-mediated activation of the HIV-1 promoter depends upon a proteasome-associated factor, PAAF1, which dissociates 26S proteasome to produce 19S RP that is essential for transcriptional elongation. The effect of PAAF1 on proteasome activity could also potentially shield certain factors from proteolysis, which may be implicated in the transcriptional co-activator activity of PAAF1 towards the LTR. RESULTS: Here, we show that Spt6 is targeted by proteasome in the absence of PAAF1. PAAF1 interacts with the N-terminus of Spt6, suggesting that PAAF1 protects Spt6 from proteolysis. Depletion of either PAAF1 or Spt6 reduced histone occupancy at the HIV-1 promoter, and induced the synthesis of aberrant transcripts. Ectopic Spt6 expression or treatment with proteasome inhibitor partially rescued the transcription defect associated with loss of PAAF1. Transcriptional profiling followed by ChIP identified a subset of cellular genes that are regulated in a similar fashion to HIV-1 by Spt6 and/or PAAF1, including many that are involved in cancer, such as BRCA1 and BARD1. CONCLUSION: These results show that intracellular levels of Spt6 are fine-tuned by PAAF1 and proteasome, which is required for HIV-1 transcription and extends to cellular genes implicated in cancer.

Suppression of HIV-1 replication by microRNA effectors.

The rate of HIV-1 gene expression is a key step that determines the kinetics of virus spread and AIDS progression. Viral entry and gene expression were described to be the key determinants for cell permissiveness to HIV. Recent reports highlighted the involvement of miRNA in regulating HIV-1 replication post-transcriptionally. In this study we explored the role of cellular factors required for miRNA-mediated mRNA translational inhibition in regulating HIV-1 gene expression. Here we show that HIV-1 mRNAs associate and co-localize with components of the RNA Induced Silencing Complex (RISC), and we characterize some of the proteins required for miRNA-mediated silencing (miRNA effectors). RCK/p54, GW182, LSm-1 and XRN1 negatively regulate HIV-1 gene expression by preventing viral mRNA association with polysomes. Interestingly, knockdown of RCK/p54 or DGCR8 resulted in virus reactivation in PBMCs isolated from HIV infected patients treated with suppressive HAART.

The proteasome regulates HIV-1 transcription by both proteolytic and nonproteolytic mechanisms.

Although the proteasome facilitates transcription from several yeast promoters, it is unclear if its role is proteolytic or which subunits are involved. We show that the proteasome regulates the HIV-1 promoter in both proteolytic and nonproteolytic modes. In the absence of transcription factor, Tat, proteasome was associated with promoter and coding regions, and its proteolytic activity regulated the level of basal transcription emanating from the promoter. Tat switched the proteasome to a nonproteolytic mode by recruiting a proteasome-associated protein, PAAF1, which favors proteasome dissociation into 19S and 20S particles. Gel filtration chromatography showed that expression of both Tat and PAAF1 enhanced the abundance of a 19S-like complex in nuclear extracts. 19S, but not 20S, subunits were strongly recruited to the promoter in the presence of Tat and PAAF1 and coactivated Tat-dependent transcription. 19S components facilitated transcriptional elongation and may be involved in clearance of paused transcriptional elongation complexes from the promoter.

Intrinsic ubiquitination activity of PCAF controls the stability of the oncoprotein Hdm2.

The p300-CBP-associated factor (PCAF) is a histone acetyltransferase (HAT) involved in the reversible acetylation of various transcriptional regulators, including the tumour suppressor p53. It is implicated in many cellular processes, such as transcription, differentiation, proliferation and apoptosis. We observed that knockdown of PCAF expression in HeLa or U2OS cell lines induces stabilization of the oncoprotein Hdm2, a RING finger E3 ligase primarily known for its role in controlling p53 stability. To investigate the molecular basis of this effect, we examined whether PCAF is involved in Hdm2 ubiquitination. Here, we show that PCAF, in addition to its acetyltransferase activity, possesses an intrinsic ubiquitination activity that is critical for controlling Hdm2 expression levels, and thus p53 functions. Our data highlight a regulatory crosstalk between PCAF and Hdm2 activities, which is likely to have a central role in the subtle control of p53 activity after DNA damage.