The histone chaperone ANP32B regulates chromatin incorporation of the atypical human histone variant macroH2A

All vertebrate genomes encode for three large histone H2A variants that have an additional metabolite-binding globular macrodomain module, macroH2A. MacroH2A variants impact heterochromatin organization and transcription regulation and establish a barrier for cellular reprogramming. However, the mechanisms of how macroH2A is incorporated into chromatin and the identity of any chaperones required for histone deposition remain elusive. Here, we develop a split-GFP-based assay for chromatin incorporation and use it to conduct a genome-wide mutagenesis screen in haploid human cells to identify proteins that regulate macroH2A dynamics. We show that the histone chaperone ANP32B is a regulator of macroH2A deposition. ANP32B associates with macroH2A in cells and in vitro binds to histones with low nanomolar affinity. In vitro nucleosome assembly assays show that ANP32B stimulates deposition of macroH2A-H2B and not of H2A-H2B onto tetrasomes. In cells, depletion of ANP32B strongly affects global macroH2A chromatin incorporation, revealing ANP32B as a macroH2A histone chaperone

CAF-1 deposits newly synthesized histones during DNA replication using distinct mechanisms on the leading and lagging strands

During every cell cycle, both the genome and the associated chromatin must be accurately replicated. Chromatin Assembly Factor-1 (CAF-1) is a key regulator of chromatin replication, but how CAF-1 functions in relation to the DNA replication machinery is unknown. Here, we reveal that this crosstalk differs between the leading and lagging strand at replication forks. Using biochemical reconstitutions, we show that DNA and histones promote CAF-1 recruitment to its binding partner PCNA and reveal that two CAF-1 complexes are required for efficient nucleosome assembly under these conditions. Remarkably, in the context of the replisome, CAF-1 competes with the leading strand DNA polymerase epsilon (Polϵ) for PCNA binding. However, CAF-1 does not affect the activity of the lagging strand DNA polymerase Delta (Polδ). Yet, in cells, CAF-1 deposits newly synthesized histones equally on both daughter strands. Thus, on the leading strand, chromatin assembly by CAF-1 cannot occur simultaneously to DNA synthesis, while on the lagging strand these processes may be coupled. We propose that these differences may facilitate distinct parental histone recycling mechanisms and accommodate the inherent asymmetry of DNA replication

CAF-1 deposits newly synthesized histones during DNA replication using distinct mechanisms on the leading and lagging strands

During every cell cycle, both the genome and the associated chromatin must be accurately replicated. Chromatin Assembly Factor-1 (CAF-1) is a key regulator of chromatin replication, but how CAF-1 functions in relation to the DNA replication machinery is unknown. Here, we reveal that this crosstalk differs between the leading and lagging strand at replication forks. Using biochemical reconstitutions, we show that DNA and histones promote CAF-1 recruitment to its binding partner PCNA and reveal that two CAF-1 complexes are required for efficient nucleosome assembly under these conditions. Remarkably, in the context of the replisome, CAF-1 competes with the leading strand DNA polymerase epsilon (Polϵ) for PCNA binding. However, CAF-1 does not affect the activity of the lagging strand DNA polymerase Delta (Polδ). Yet, in cells, CAF-1 deposits newly synthesized histones equally on both daughter strands. Thus, on the leading strand, chromatin assembly by CAF-1 cannot occur simultaneously to DNA synthesis, while on the lagging strand these processes may be coupled. We propose that these differences may facilitate distinct parental histone recycling mechanisms and accommodate the inherent asymmetry of DNA replication

One Chaperone to Rule Them All: Deciphering How Chromatin is Assembled During DNA Replication

Genomic DNA, which governs cellular life, resides within the nucleus of every human cell. Inside each nucleus lies approximately two meters of DNA, posing a significant challenge, akin to compacting 20 kilometers of thread into a tennis ball. To overcome this challenge, DNA is packaged by wrapping itself around proteins called histones, forming individual structures known as nucleosomes. This succession of nucleosomes creates an organization that resembles beads on string, commonly referred to as chromatin. Histones not only serve as a structural framework for the chromatin; they also regulate its accessibility by carrying chemical modifications that directly impact vital cellular processes. Preserving this chromatin information, also referred to as epigenetic information, is crucial to ensuring the survival of each cell and, by extension, the organism. Throughout life, many cell types in the human body undergo cell divisions (like skin cells for instance). However, before any cell division can occur, the cell must replicate its genetic content to provide an intact copy of the genome to the future two daughter cells. This process is known as DNA replication. Alongside DNA replication, epigenetic information on histones is meticulously reconstituted, via a process called chromatin replication. Failure in chromatin replication would severely affect the function and identity of the future daughter cells. For instance, a skin cell that failed to replicate its chromatin before dividing, would give rise to two daughter cells that are no longer skin cells. Chromatin replication involves the wrapping of DNA around histones to form chromatin. To ensure the smooth progress of this operation, histone proteins, known for their instability and propensity for aggregation, require careful handling. Histone chaperones, a class of proteins, take charge of protecting histones, ensuring their controlled trafficking, and facilitating their deposition onto the DNA. In the context of chromatin replication, the histone chaperone Chromatin Assembly Factor 1 (CAF-1) plays a central role by directly depositing histones onto the DNA. CAF-1 is precisely targeted to DNA synthesis sites through direct interaction with the protein PCNA. The work presented in this thesis explores the complex coordination between CAF-1 and PCNA in chromatin replication by examining the dynamics of their interaction during DNA synthesis

NMR assignment and solution structure of the external DII domain of the yeast Rvb2 protein

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The histone chaperone ANP32B regulates chromatin incorporation of the atypical human histone variant macroH2A

We would like to thank Carla Margulies, all members of the Ladurner and Mattiroli labs, and the Department of Physiological Chemistry for productive discussions. Thanks to Jan Dreyer, Inge Rondeel, and Rosanne van Hooijdonk for assistance with experiments. We thank the Bioimaging, Biophysics, and Flow Cytometry Core Facilities of the LMU Biomedical Center for training and use of their resources. We acknowledge the Protein Analytics Unit at the Biomedical Center, Ludwig-Maximilians University Munich (DFG, RI-00089), for providing services and assistance with data analysis. We thank the Histone Source at Colorado State University for the purification of human histones. pCAGGS-ANP32B was a kind gift from Wendy Barclay and pET29a-YS14 was a kind gift from Jung-Hyun Min (Addgene plasmid # 66890). pQCXIP-GFP1-10 was a gift from Yutaka Hata (Addgene plasmid # 68715) and pRK-flag-GFP11 from Yihong Ye (Addgene plasmid # 78590). This work was supported by funding from the Dutch Research Council (VI.Veni.212.052 to I.K.M.), the European Commission (ERC StG 851564 to F.M.; ERC StG 804182 to L.T.J.), the DFG (German Research Foundation) through Project-ID 213249687 - SFB 1064 and Project-ID 325871075 - SFB 1309, as well as LMU (to A.G.L.) and the national grant PID2021-126907NB-I00 from FEDER/Ministerio de Ciencia e Innovación (MCIN) - Agencia Estatal de Investigación and the Fundació La Marató de TV3 257/C/2019 (to M.B.).We would like to thank Carla Margulies, all members of the Ladurner and Mattiroli labs, and the Department of Physiological Chemistry for productive discussions. Thanks to Jan Dreyer, Inge Rondeel, and Rosanne van Hooijdonk for assistance with experiments. We thank the Bioimaging, Biophysics, and Flow Cytometry Core Facilities of the LMU Biomedical Center for training and use of their resources. We acknowledge the Protein Analytics Unit at the Biomedical Center, Ludwig-Maximilians University Munich (DFG, RI-00089), for providing services and assistance with data analysis. We thank the Histone Source at Colorado State University for the purification of human histones. pCAGGS-ANP32B was a kind gift from Wendy Barclay and pET29a-YS14 was a kind gift from Jung-Hyun Min (Addgene plasmid # 66890). pQCXIP-GFP1-10 was a gift from Yutaka Hata (Addgene plasmid # 68715) and pRK-flag-GFP11 from Yihong Ye (Addgene plasmid # 78590). This work was supported by funding from the Dutch Research Council (VI.Veni.212.052 to I.K.M.), the European Commission (ERC StG 851564 to F.M.; ERC StG 804182 to L.T.J.), the DFG (German Research Foundation) through Project-ID 213249687 - SFB 1064 and Project-ID 325871075 - SFB 1309, as well as LMU (to A.G.L.) and the national grant PID2021-126907NB-I00 from FEDER/Ministerio de Ciencia e Innovación (MCIN) - Agencia Estatal de Investigación and the Fundació La Marató de TV3 257/C/2019 (to M.B.). Conceptualization: I.K.M. and A.G.L.; methodology: I.K.M. C.R. F.M. E.F. and L.T.J.; investigation: I.K.M. E.F. D.C. and C.K.; resources: C.R.; writing - original draft: I.K.M.; writing - review and editing: F.M. and A.G.L.; supervision: L.T.J. F.M. M.B. and A.G.L.; funding acquisition: I.K.M. L.T.J. M.B. F.M. and A.G.L. A.G.L. is a founder, CSO, and managing director of Eisbach Bio GmbH, a biotechnology company in oncology and virology. We support inclusive, diverse, and equitable conduct of research.All vertebrate genomes encode for three large histone H2A variants that have an additional metabolite-binding globular macrodomain module, macroH2A. MacroH2A variants impact heterochromatin organization and transcription regulation and establish a barrier for cellular reprogramming. However, the mechanisms of how macroH2A is incorporated into chromatin and the identity of any chaperones required for histone deposition remain elusive. Here, we develop a split-GFP-based assay for chromatin incorporation and use it to conduct a genome-wide mutagenesis screen in haploid human cells to identify proteins that regulate macroH2A dynamics. We show that the histone chaperone ANP32B is a regulator of macroH2A deposition. ANP32B associates with macroH2A in cells and in vitro binds to histones with low nanomolar affinity. In vitro nucleosome assembly assays show that ANP32B stimulates deposition of macroH2A-H2B and not of H2A-H2B onto tetrasomes. In cells, depletion of ANP32B strongly affects global macroH2A chromatin incorporation, revealing ANP32B as a macroH2A histone chaperone

CAF-1 deposits newly synthesized histones during DNA replication using distinct mechanisms on the leading and lagging strands

During every cell cycle, both the genome and the associated chromatin must be accurately replicated. Chromatin Assembly Factor-1 (CAF-1) is a key regulator of chromatin replication, but how CAF-1 functions in relation to the DNA replication machinery is unknown. Here, we reveal that this crosstalk differs between the leading and lagging strand at replication forks. Using biochemical reconstitutions, we show that DNA and histones promote CAF-1 recruitment to its binding partner PCNA and reveal that two CAF-1 complexes are required for efficient nucleosome assembly under these conditions. Remarkably, in the context of the replisome, CAF-1 competes with the leading strand DNA polymerase epsilon (Polϵ) for PCNA binding. However, CAF-1 does not affect the activity of the lagging strand DNA polymerase Delta (Polδ). Yet, in cells, CAF-1 deposits newly synthesized histones equally on both daughter strands. Thus, on the leading strand, chromatin assembly by CAF-1 cannot occur simultaneously to DNA synthesis, while on the lagging strand these processes may be coupled. We propose that these differences may facilitate distinct parental histone recycling mechanisms and accommodate the inherent asymmetry of DNA replication.</p