Highlights d Marseilleviridae encode proteins that resemble fused histones H4-H3 and H2B-H2A d These histone doublets assemble into unstable nucleosomelike particles in vitro d Histone doublets localize to the viral factory and are highly abundant in the virus d They are essential for viral fitness and infectivity, a first for any virus

Recent integrative epigenome analyses highlight the importance of functionally distinct chromatin states for accurate cell function. How these states are established and maintained is a matter of intense investigation. Here, we present evidence for DNA damage as an unexpected means to shape a protective chromatin environment at regions of recurrent replication stress (RS). Upon aberrant fork stalling, DNA damage signaling and concomitant H2AX phosphorylation coordinate the FACT-dependent deposition of macroH2A1.2, a histone variant that promotes DNA repair by homologous recombination (HR). MacroH2A1.2, in turn, facilitates the accumulation of the tumor suppressor and HR effector BRCA1 at replication forks to protect from RS-induced DNA damage. Consequently, replicating primary cells steadily accrue macroH2A1.2 at fragile regions, whereas macroH2A1.2 loss in these cells triggers DNA damage signaling-dependent senescence, a hallmark of RS. Altogether, our findings demonstrate that recurrent DNA damage contributes to the chromatin landscape to ensure the epigenomic integrity of dividing cells.

The histone chaperone FACT functions by tethering partial components of the nucleosome, thereby assisting nucleosome disassembly and reassembly during transcription.

Human FACT (hFACT) is a conserved histone chaperone that was originally described as a transcription elongation factor with potential nucleosome assembly functions. Here we show that FACT facilitates tetrasome assembly and H2A-H2B deposition to form hexasomes and nucleosomes. In the process, FACT tethers components of the nucleosome through interactions with H2A-H2B, resulting in a defined intermediate complex comprised of FACT, a histone hexamer and DNA. Free DNA extending from the tetrasome then competes FACT off H2A-H2B, thereby promoting hexasome and nucleosome formation. Our studies provide mechanistic insight into how FACT may stabilize partial nucleosome structures during transcription or nucleosome assembly, seemingly facilitating nucleosome disassembly and nucleosome assembly.

The histone variant CENP-A is the epigenetic determinant for the centromere, where it is interspersed with canonical H3 to form a specialized chromatin structure that nucleates the kinetochore. How nucleosomes at the centromere arrange into higher order structures is unknown. Here we demonstrate that the human CENP-A-interacting protein CENP-N promotes the stacking of CENP-A-containing mononucleosomes and nucleosomal arrays through a previously undefined interaction between the α6 helix of CENP-N with the DNA of a neighboring nucleosome. We describe the cryo-EM structures and biophysical characterization of such CENP-N-mediated nucleosome stacks and nucleosomal arrays and demonstrate that this interaction is responsible for the formation of densely packed chromatin at the centromere in the cell. Our results provide first evidence that CENP-A, together with CENP-N, promotes specific chromatin higher order structure at the centromere.