AbstractThe histone variant macroH2A1 (mH2A1) is involved in cellular growth, differentiation and reprogramming, but the underlying molecular mechanisms are a matter of debate. Different roles of mH2A1 in gene expression may relate to functional differences of its two splicing isoforms, mH2A1.1 and mH2A1.2. Here, we map for the first time genome-wide localization of endogenous mH2A1.1 and link the distribution of mH2A1.1 to control of gene expression in human breast cancer cells. In addition to localization shared with mH2A1.2 to facultative heterochromatin, mH2A1.1 specifically associates with regulatory elements required for gene activation, super-enhancers and promoters of highly expressed genes. Depending on the recruitment profile of mH2A1.1 to these elements, selective depletion of mH2A1.1 up- or downregulates its target genes. mH2A1.1 represses transcription when its binding is spread over the entire gene and promoter, and activates transcription when its binding is strictly confined to the transcription start site (TSS). Notably, RNA Polymerase II was frequently in pause at mH2A1.1-activated genes. Functionally, mH2A1.1-dependent regulation of a subset of paused genes impedes mammary tumor cell migration. Molecular mechanisms of mH2A1.1 function at the TSS uncovered by our study define an intriguing new mode of transcription regulation in cancer cells.Author SummaryControl of gene expression driving cellular functions from differentiation to epistasis and causing, when dysfunctional, uncountable diseases, relies on modifications of chromatin structure. One key element enabling chromatin plasticity is the replacement of canonical histones by histone variants. Among histone variants macroH2A1 (mH2A) is an extraordinary H2A variant possessing a large non-histone domain placed outside of the nucleosome. Two splicing isoforms, mH2A1.1 and mH2A1.2, are produced, but these are rarely studied separately because they only differ in a 30 amino acid region and are difficult to distinguish experimentally, which likely explains contradictory functions reported in the literature. Here, we take advantage of a mH2A1.1 specific antibody to generate the first genome-wide chromatin-associated map of this histone variant in the invasive breast cancer cells line MDA-MB231. We confirm that mH2A1.1, like mH2A1.2, is enriched at facultative heterochromatin in agreement with its reported role as a repressor. However, we discovered that unlike its splicing isoform, mH2A1.1 specifically binds to super-enhancers and the transcription start site of highly transcribed genes. mH2A1.1 is necessary for regulating transcription of these genes. At the cellular level, we demonstrate that mH2A1.1 inhibits migration capacity of highly metastatic breast cancer cells. Our study characterizes for the first time binding profiles of mH2A1.1 that are linked to regulation of gene expression, thereby providing a new molecular mechanisms which govern the plasticity of human tumor cells.