Otto Hudecz

Genomic DNA is assembled into chromatin by histones, and extruded into loops by cohesin. These mechanisms control important genomic functions, but whether histones and cohesin cooperate in genome regulation is poorly understood. Here we identify Phf2, a member of the Jumonji-C family of histone demethylases, as a cohesin-interacting protein. Phf2 binds to H3K4me3 nucleosomes at active transcription start sites (TSSs), but also co-localizes with cohesin. Cohesin depletion reduces Phf2 binding at sites lacking H3K4me3, and depletion of Wapl and CTCF re-positions Phf2 together with cohesin in the genome, resulting in the accumulation of both proteins in chromosomal regions called vermicelli and cohesin islands. Conversely, Phf2 depletion reduces cohesin binding at TSSs lacking CTCF and decreases the number of short cohesin loops, while increasing the length of heterochromatic B compartments. These results suggest that Phf2 is an ‘epigenetic reader’, which is translocated through the genome by cohesin-mediated DNA loop extrusion, and which recruits cohesin to active TSSs and limits the size of B compartments. These findings reveal an unexpected degree of cooperativity between epigenetic and architectural mechanisms of eukaryotic genome regulation.

Inhibitor of apoptosis proteins (IAPs) bind to pro-apoptotic proteases, keeping them inactive and preventing cell death. The atypical ubiquitin ligase BIRC6 is the only essential IAP, additionally functioning as a suppressor of autophagy. We performed a structure-function analysis of BIRC6 in complex with caspase-9, HTRA2, SMAC, and LC3B, which are critical apoptosis and autophagy proteins. Cryo–electron microscopy structures showed that BIRC6 forms a megadalton crescent shape that arcs around a spacious cavity containing receptor sites for client proteins. Multivalent binding of SMAC obstructs client binding, impeding ubiquitination of both autophagy and apoptotic substrates. On the basis of these data, we discuss how the BIRC6/SMAC complex can act as a stress-induced hub to regulate apoptosis and autophagy drivers.

Summary: Inhibitor of apoptosis proteins (IAPs) bind to pro-apoptotic proteases, keeping them inactive and preventing cell death. BIRC6 is an exceptionally large, multidomain IAP that inhibits its targets by means of its atypical ubiquitin ligase activity and in addition, functions as an inhibitor of autophagy by depleting LC3B. Little is known of the mechanisms by which BIRC6 interacts with its targets and fulfills these two roles. Here, we determined the cryo-EM structure of BIRC6 alone and in complex with two mitochondrial pro-apoptotic proteins, HTRA2 and SMAC. We show BIRC6 is an antiparallel homodimer that forms a crescent shape that arcs around a spacious cavity. The cavity is surrounded by binding sites for client proteins, where they interact with the flexible UBC domain that mediates ubiquitin ligation. Functional data reveal that multivalent binding of SMAC in the central cavity obstructs substrate binding, impeding ubiquitination of both autophagy and apoptotic target proteins. Together our data reveal the molecular mechanisms of how SMAC specifically binds and inhibits BIRC6 to promote apoptosis, and how this regulatory mechanism also extends to autophagy substrates. The interaction sites are hot spots of cancer and atrophy mutations, highlighting the importance of carefully balancing the interplay between BIRC6 and SMAC.