The histone variant macroH2A1.1 regulates RNA polymerase II-paused genes within defined chromatin interaction landscapes.

The histone variant macroH2A1.1 plays a role in cancer development and metastasis. To determine the underlying molecular mechanisms, we mapped the genome-wide localization of endogenous macroH2A1.1 in the human breast cancer cell line MDA-MB-231. We demonstrate that macroH2A1.1 specifically binds to active promoters and enhancers in addition to facultative heterochromatin. Selective knock down of macroH2A1.1 deregulates the expression of hundreds of highly active genes. Depending on the chromatin landscape, macroH2A1.1 acts through two distinct molecular mechanisms. The first mitigates excessive transcription by binding over domains including the promoter and the gene body. The second stimulates expression of RNA polymerase II (Pol II)-paused genes, including genes regulating mammary tumor cell migration. In contrast to the first mechanism, macroH2A1.1 specifically associates with the transcription start site of Pol II-paused genes. These processes occur in a predefined local 3D genome landscape, but do not require rewiring of enhancer-promoter contacts. We thus propose that macroH2A1.1 serves as a transcriptional modulator with a potential role in assisting the conversion of promoter-locked Pol II into a productive, elongating Pol II.

Recruitment of the Histone Variant MacroH2A1 to the Pericentric Region Occurs upon Chromatin Relaxation and Is Responsible for Major Satellite Transcriptional Regulation.

Heterochromatin formation plays a pivotal role in regulating chromatin organization and influences nuclear architecture and genome stability and expression. Amongst the locations where heterochromatin is found, the pericentric regions have the capability to attract the histone variant macroH2A1. However, the factors and mechanisms behind macroH2A1 incorporation into these regions have not been explored. In this study, we probe different conditions that lead to the recruitment of macroH2A1 to pericentromeric regions and elucidate its underlying functions. Through experiments conducted on murine fibroblastic cells, we determine that partial chromatin relaxation resulting from DNA damage, senescence, or histone hyper-acetylation is necessary for the recruitment of macroH2A1 to pericentric regions. Furthermore, macroH2A1 is required for upregulation of noncoding pericentric RNA expression but not for pericentric chromatin organization. Our findings shed light on the functional rather than structural significance of macroH2A1 incorporation into pericentric chromatin.

The THAP-zinc finger protein THAP1 associates with coactivator HCF-1 and O-GlcNAc transferase: a link between DYT6 and DYT3 dystonias.

THAP1 is a sequence-specific DNA binding factor that regulates cell proliferation through modulation of target genes such as the cell cycle-specific gene RRM1. Mutations in the THAP1 DNA binding domain, an atypical zinc finger (THAP-zf), have recently been found to cause DYT6 dystonia, a neurological disease characterized by twisting movements and abnormal postures. In this study, we report that THAP1 shares sequence characteristics, in vivo expression patterns and protein partners with THAP3, another THAP-zf protein. Proteomic analyses identified HCF-1, a potent transcriptional coactivator and cell cycle regulator, and O-GlcNAc transferase (OGT), the enzyme that catalyzes the addition of O-GlcNAc, as major cellular partners of THAP3. THAP3 interacts with HCF-1 through a consensus HCF-1-binding motif (HBM), a motif that is also present in THAP1. Accordingly, THAP1 was found to bind HCF-1 in vitro and to associate with HCF-1 and OGT in vivo. THAP1 and THAP3 belong to a large family of HCF-1 binding factors since seven other members of the human THAP-zf protein family were identified, which harbor evolutionary conserved HBMs and bind to HCF-1. Chromatin immunoprecipitation (ChIP) assays and RNA interference experiments showed that endogenous THAP1 mediates the recruitment of HCF-1 to the RRM1 promoter during endothelial cell proliferation and that HCF-1 is essential for transcriptional activation of RRM1. Together, our findings suggest HCF-1 is an important cofactor for THAP1. Interestingly, our results also provide an unexpected link between DYT6 and DYT3 (X-linked dystonia-parkinsonism) dystonias because the gene encoding the THAP1/DYT6 protein partner OGT maps within the DYT3 critical region on Xq13.1.

Analysis of Cellular EMT States Using Molecular Biology and High Resolution FISH Labeling.

Metastasis results from the ability of cancer cells to grow and to spread beyond the primary tumor to distant organs. Epithelial-to-Mesenchymal Transition (EMT), a fundamental developmental process, is reactivated in cancer cells, and causes epithelial properties to evolve into mesenchymal and invasive ones. EMT changes cellular characteristics between two distinct states, yet, the process is not binary but rather reflects a broad spectrum of partial EMT states in which cells exhibit various degrees of intermediate epithelial and mesenchymal phenotypes. EMT is a complex multistep process that involves cellular reprogramming through numerous signaling pathways, alterations in gene expression, and changes in chromatin morphology. Therefore, expression of key proteins, including cadherins, occludin, or vimentin must be precisely regulated. A comprehensive understanding of how changes in nuclear organization, at the level of single genes clusters, correlates with these processes during formation of metastatic cells is still missing and yet may help personalized prognosis and treatment in the clinic. Here, we describe methods to correlate physiological and molecular states of cells undergoing an EMT process with chromatin rearrangements observed via FISH labeling of specific domains.

The Histone Variant MacroH2A1 Regulates Key Genes for Myogenic Cell Fusion in a Splice-Isoform Dependent Manner.

MacroH2A histone variants have functions in differentiation, somatic cell reprogramming and cancer. However, at present, it is not clear how macroH2As affect gene regulation to exert these functions. We have parted from the initial observation that loss of total macroH2A1 led to a change in the morphology of murine myotubes differentiated ex vivo. The fusion of myoblasts to myotubes is a key process in embryonic myogenesis and highly relevant for muscle regeneration after acute or chronic injury. We have focused on this physiological process, to investigate the functions of the two splice isoforms of macroH2A1. Individual perturbation of the two isoforms in myotubes forming in vitro from myogenic C2C12 cells showed an opposing phenotype, with macroH2A1.1 enhancing, and macroH2A1.2 reducing, fusion. Differential regulation of a subset of fusion-related genes encoding components of the extracellular matrix and cell surface receptors for adhesion correlated with these phenotypes. We describe, for the first time, splice isoform-specific phenotypes for the histone variant macroH2A1 in a physiologic process and provide evidence for a novel underlying molecular mechanism of gene regulation.

The THAP domain: a novel protein motif with similarity to the DNA-binding domain of P element transposase.

We have identified a novel evolutionarily conserved protein motif - designated the THAP domain - that defines a new family of cellular factors. We have found that the THAP domain presents striking similarities with the site-specific DNA-binding domain (DBD) of Drosophila P element transposase, including a similar size, N-terminal location, and conservation of the residues that define the THAP motif, such as the C2CH signature (Cys-Xaa(2-4)-Cys-Xaa(35-50)-Cys-Xaa(2)-His). Our results suggest that the THAP domain is a novel example of a DBD that is shared between cellular proteins and transposases from mobile genomic parasites.

Increased macroH2A1.1 expression correlates with poor survival of triple-negative breast cancer patients.

PURPOSE: Epithelial-Mesenchymal Transition (EMT) features appear to be key events in development and progression of breast cancer. Epigenetic modifications contribute to the establishment and maintenance of cancer subclasses, as well as to the EMT process. Whether histone variants contribute to these transformations is not known. We investigated the relative expression levels of histone macroH2A1 splice variants and correlated it with breast cancer status/prognosis/types. METHODS: To detect differential expression of macroH2A1 variant mRNAs in breast cancer cells and tumor samples, we used the following databases: GEO, EMBL-EBI and publisher databases (may-august 2012). We extracted macroH2A1.1/macroH2A1 mRNA ratios and performed correlation studies on intrinsic molecular subclasses of breast cancer and on molecular characteristics of EMT. Associations between molecular and survival data were determined. RESULTS: We found increased macroH2A1.1/macroH2A1 mRNA ratios to be associated with the claudin-low intrinsic subtype in breast cancer cell lines. At the molecular level this association translates into a positive correlation between macroH2A1 ratios and molecular characteristics of the EMT process. Moreover, untreated Triple Negative Breast Cancers presenting a high macroH2A1.1 mRNA ratio exhibit a poor outcome. CONCLUSION: These results provide first evidence that macroH2A1.1 could be exploited as an actor in the maintenance of a transient cellular state in EMT progress towards metastatic development of breast tumors.

Exogenously added fibroblast growth factor 2 (FGF-2) to NIH3T3 cells interacts with nuclear ribosomal S6 kinase 2 (RSK2) in a cell cycle-dependent manner.

Fibroblast growth factor 2 (FGF-2) has been detected in the nuclei of many tissues and cell lines. Here we demonstrate that FGF-2 added exogenously to NIH3T3 cells enters the nucleus and interacts with the nuclear active 90-kDa ribosomal S6 kinase 2 (RSK2) in a cell cycle-dependent manner. By using purified proteins, FGF-2 is shown to directly interact through two separate domains with two RSK2 domains on both sides of the hydrophobic motif, namely the NH2-terminal kinase domain (residues 360-381) by amino acid Ser-117 and the COOH-terminal kinase domain (residues 388-400) by amino acids Leu-127 and Lys-128. Moreover, this interaction leads to maintenance of the sustained activation of RSK2 in G1 phase of the cell cycle. FGF-2 mutants (FGF-2 S117A, FGF-2 L127A, and FGF-2 K128A) that fail to interact in vitro with RSK2 fail to maintain a sustained RSK2 activity in vivo.

TAF7 (TAFII55) plays a role in the transcription activation by c-Jun.

c-Jun is a member of the AP-1 family of transcription factors regulating expression of specific target genes in a variety of cellular processes including proliferation, stress response, and tumorigenicity. In the present study we have analyzed the mechanism of c-Jun function as a transactivator with respect to members of the basal transcription machinery, TATA-binding protein-associated factors (TAFs). We show that one member of the family, human TAF7 (formerly TAFII55), physically interacts with c-Jun through two independent interaction domains, within the N- and C-terminal part of c-Jun. Interaction in vitro correlates with enhanced transactivation function of c-Jun in HEK293 and COS cells in the presence of increasing amounts of TAF7. TAF7 interacts preferentially with DNA-bound phosphorylated c-Jun, suggesting that TAF7 represents a novel c-Jun co-activator mediating activation of AP-1 target genes in response to extracellular signals.