Biology subjects

Bilokapic, S.

Publications and source records attributed to Bilokapic, S..

5 recordsLinked to original sources

bioRxiv2026

PARP1 directly disassembles nucleosomes to regulate DNA repair

Upon DNA damage, chromatin remodeling is rapidly initiated to promote chromatin accessibility, thereby facilitating the recruitment and assembly of repair factors. Although this enhanced accessibility has been linked to poly(ADP-ribose) polymerase (PARP) activity, the mechanism by which cells overcome the nucleosome barrier remains unclear. Using our designer chromatin system, we uncovered a previously uncharacterized activity of PARP1, whereby it directly and asymmetrically evicts histone dimers proximal to DNA strand breaks from nucleosomes to generate oriented hexasomes. In the presence of HPF1, PARP1 generates stable PARylated hexasomes, an open chromatin intermediate that can serve as a bifunctional hub for recruitment of DNA- and PAR-dependent factors. Using cellular assays, we demonstrated that PARP activity is both required and sufficient to drive chromatin accessibility and the recruitment of repair factors, with direct involvement of subnucleosomal species. Unexpectedly, we identified the C-terminal tail of histone H2A, a motif harboring recurrent cancer-associated mutations, as a critical determinant of efficient PARP1-mediated nucleosome disassembly. Deletion of the H2A tail sensitizes cells to DNA-damaging agents and PARP inhibitors, implicating a functional role of PARP1-mediated nucleosome disassembly in DNA repair. Together, our findings support a model in which PARP1 directly drives histone eviction, leading to the formation of subnucleosomes that facilitate efficient DNA repair.

molecular biology↗

bioRxiv2025

Mechanisms of chromatin remodeling by a Snf2-type ATPase

Chromatin remodeling enzymes play a crucial role in the organization of chromatin, enabling both stability and plasticity of genome regulation. These enzymes use a Snf2-type ATPase motor to move nucleosomes, but how they translocate DNA around the histone octamer is unclear. Here we use cryo-EM to visualize the continuous motion of nucleosomal DNA induced by human chromatin remodeler SNF2H, an ISWI family member. Our work reveals conformational changes in SNF2H, DNA and histones during nucleosome sliding and provides the structural basis for DNA translocation. ATP hydrolysis induces conformational changes in SNF2H that pull the DNA tracking strand, distorting DNA and histones at SHL2. This is followed by SNF2H rotation on the nucleosome, which first pulls the DNA guide strand and creates one-base pair bulge at SHL2, and then releases the pulled DNA. Given the high conservation of the catalytic motors among ATP-dependent chromatin remodelers, the mechanisms we describe likely apply to other families.

biophysics↗

bioRxiv2025

Non-nucleosomal (CENP-A/H4)2 - DNA complexes as a possible platform for centromere organization

The centromere is a part of the chromosome that is essential for the even segregation of duplicated chromosomes during cell division. It is epigenetically defined by the presence of the histone H3 variant CENP-A. CENP-A associates specifically with a group of 16 proteins that form the centromere-associated network of proteins (CCAN). In mitosis, the kinetochore forms on the CCAN to connect the duplicated chromosomes to the microtubules protruding from the cell poles. Previous studies have shown that CENP-A replaces H3 in nucleosomes, and recently the structures of CENP-A-containing nucleosomes in complex with CCANs have been revealed, but they show only a limited interaction between CCANs and CENP-A. Here, we report the cryoEM structure of 2x(CENP-A/H4)2-di-tetramers assembled on DNA in the absence of H2A/H2B histone dimer and speculate how (CENP-A/H4)2-tetramers and -di-tetramers might serve as a platform for CCAN organization. Main pointsO_LICENP-A/H4 in the absence of H2A/H2B dimers associate on DNA to form di-tetrasomes C_LIO_LICENP-A/H4 di-tetrasomes are stable but highly flexible structures resembling nucleosomes C_LIO_LICENP-A/H4 tetrasomes bind CENP-N/L and can serve as platform for centromere organisation C_LI

biochemistry↗

bioRxiv2023

Histone modifications regulate pioneer transcription factor cooperativity

Pioneer transcription factors have the ability to access DNA in compacted chromatin. Multiple transcription factors can bind together to a regulatory element in a cooperative way and cooperation between pioneer transcription factors Oct4 and Sox2 is important for pluripotency and reprogramming. However, the molecular mechanisms by which pioneer transcription factors function and cooperate remain unclear. Here we present cryo-EM structures of human Oct4 bound to a nucleosome containing human Lin28B and nMatn1 DNA sequences, which bear multiple binding sites for Oct4. Our structural and biochemistry data reveal that Oct4 binding induces changes to the nucleosome structure, repositions the nucleosomal DNA and facilitates cooperative binding of additional Oct4 and of Sox2 to their internal binding sites. The flexible activation domain of Oct4 contacts the histone H4 N-terminal tail, altering its conformation and thus promoting chromatin decompaction. Moreover, the DNA binding domain of Oct4 engages with histone H3 N-terminal tail, and posttranslational modifications at H3K27 modulate DNA positioning and affect transcription factor cooperativity. Thus, our data show that the epigenetic landscape can regulate Oct4 activity to ensure proper cell reprogramming.

biophysics↗

bioRxiv2019

CryoEM structure of human CENP-A nucleosome alone and in complex with central domain of human CENP-C

Centromeres are defined epigenetically by nucleosomes containing the histone H3 variant CENP-A, upon which the constitutive centromere-associated network of proteins (CCAN) is built. CENP-C, is considered to be a central organizer of the CCAN. We provide new molecular insights into the structure of CENP-A nucleosomes, in isolation and in complex with the CENP-C central region (CENP-CCR), the main CENP-A binding module of CENP-C. We establish that the short N-helix of CENP-A promotes DNA flexibility at the nucleosome ends, independently of the sequence it wraps.\n\nFurthermore, we show that, in vitro, two regions of CENP-C (CENP-CCR and CENP-Cmotif) both bind exclusively to the CENP-A nucleosome. We find CENP-CCR to bind with high affinity due to an extended hydrophobic area made up of CENP-AV532 and CENP-AV533. Importantly, we identify two key conformational changes within the CENP-A nucleosome upon CENP-C binding. First, the loose DNA wrapping of CENP-A nucleosomes is further exacerbated, through destabilization of the H2A N-terminal tail. Second, CENP-CCR rigidifies the N-terminal tail of H4 in the conformation favoring H4K20 monomethylation, essential for a functional centromere.\n\nSynopsisCENP-A nucleosomes have a short N helix incompatible with complete DNA wrapping, independently of DNA sequence. CENP-C binds exclusively to CENP-A nucleosomes and this binding induces conformational changes that further differentiate CENP-A-containing from canonical nucleosomes.\n\nO_LICENP-C binds CENP-A nucleosomes specifically\nC_LIO_LIDNA ends of the CENP-A nucleosome are further unwrapped in the CENP-A/CENP-C complex, due to flexible H2A C-terminal tails\nC_LIO_LIThe N-terminal tail of H4 adopts a conformation favored for centromere specific H4K20 monomethylation when CENP-C is bound\nC_LI

biochemistry↗