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.

HDAC inhibition does not induce estrogen receptor in human triple-negative breast cancer cell lines and patient-derived xenografts.

Several publications have suggested that histone deacetylase inhibitors (HDACis) could reverse the repression of estrogen receptor alpha (ERα) in triple-negative breast cancer (TNBC) cell lines, leading to the induction of a functional protein. Using different HDACis, vorinostat, panobinostat, and abexinostat, we therefore investigated this hypothesis in various human TNBC cell lines and patient-derived xenografts (PDXs). We used three human TNBC cell lines and three PDXs. We analyzed the in vitro toxicity of the compounds, their effects on the hormone receptors and hormone-related genes and protein expression both in vitro and in vivo models. We then explored intra-tumor histone H3 acetylation under abexinostat in xenograft models. Despite major cytotoxicity of all tested HDAC inhibitors and repression of deactylation-dependent CCND1 gene, neither ERα nor ERß, ESR1 or ESR2 genes respectively, were re-expressed in vitro. In vivo, after administration of abexinostat for three consecutive days, we did not observe any induction of ESR1 or ESR1-related genes and ERα protein expression by RT-qPCR and immunohistochemical methods in PDXs. This observation was concomitant to the fact that in vivo administration of abexinostat increased intra-tumor histone H3 acetylation. These observations do not allow us to confirm previous studies which suggested that HDACis are able to convert ER-negative (ER-) tumors to ER-positive (ER+) tumors, and that a combination of HDAC inhibitors and hormone therapy could be proposed in the management of TNBC patients.

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.

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.

Activation of p21 by HDAC inhibitors requires acetylation of H2A.Z.

Differential positioning of the histone variant H2A.Z in a p53 dependent manner was shown to regulate p21 transcription. Whether H2A.Z is involved in p21 activity in the absence of p53 is not known. The p21 gene is repressed in estrogen receptor (ER) negative cell lines that are p53-/- and hormone independent for their growth. Here we demonstrate that class I and II pan Histone deacetylase inhibitors (HDACi) induce p21 transcription and reduce cell proliferation of MDA-MB231, an ERα-negative mammary tumor cell line, in a H2A.Z dependent manner. H2A.Z is associated with the transcription start site (TSS) of the repressed p21 gene. Depleting H2A.Z did not lead to transcription of p21 but annihilated the stimulating effect of HDACi on this gene. Acetylation of H2A.Z but not of H3K9 at the p21 promoter correlated with p21 activation. We further show that HDACi treatment reduced the presence of the p400 chromatin remodeler at the p21 TSS. We propose a model in which association of p400 negatively affects p21 transcription by interfering with acetylation of H2A.Z.