MacroH2A is a histone variant with a tripartite structure, consisting of a histone fold, a lysine-rich linker, and a macrodomain, making it three times larger than the canonical histones. MacroH2A and its isoforms have been implicated in a wide array of biological processes, including genome organization, heterochromatin maintenance, senescence, tumour suppression, DNA damage response, and gene repression. However, the mechanisms by which macroH2A exerts its functions, especially across its diverse range of roles, remain largely speculative. For example, the loss of macroH2A has been associated with heterochromatin depletion and nuclear lamina deformation, suggesting a role in lamina tethering or heterochromatin maintenance. Our findings reveal that in RPE-1 hTERT cells (and other cell lines), macroH2A localizes not only to lamina-associated domains but also to transcriptionally active regions of the genome. Interestingly, knockout of macroH2A1, the predominant isoform expressed in RPE-1 hTERT cells, did not affect chromatin fibre structure, chromatin accessibility, or gene expression in these cells. However, upon TGFB1 stimulation, a subset of genes are upregulated in macroH2A1 knockout cells, emphasising macroH2A1's role as a barrier against transcriptional pre-programming.