Linker histone structure, function, and dynamics in Xenopus egg extracts and embryos

Compaction of genomic DNA is essential for cellular function: to fit billions of base pairs into a tiny nucleus, to establish and maintain gene expression patterns, and to tightly package mitotic chromosomes for accurate segregation during cell division. DNA is packaged by core histones into repeating nucleosome subunits, yielding a chromatin fiber with an intrinsic capacity to fold and unfold. H1 histones, which feature a globular DNA-binding domain and a long, unstructured tail, stabilize chromatin's folded state, which is further compacted into higher-order conformations by various chromatin-associated proteins. Xenopus egg extracts provide a powerful system in which to study chromatin structure and function. Extracts can recapitulate the cell cycle in a test tube, remodeling exogenous DNA first into replicating interphase nuclei and later into mitotic chromosomes capable of segregation. Biochemical manipulation of these extracts makes possible the study of fundamental chromatin components on these organelle-scale, physiological DNA structures.;In lower-order chromatin structure, the wrapping of DNA around core histone octamers is well-established, but how histone H1 compacts chromatin is poorly understood. A current view of H1 function is that it a highly dynamic component of chromatin whose binding neutralizes repulsive charges on linker DNA and nucleosomes and thereby stabilizes the folded conformation. H1 stabilizes purified chromatin in hypotonic buffer in vitro, but since H1 is difficult to knock out genetically, it is still unknown how H1 affects structures such as nuclei and mitotic chromosomes in vivo. Functional analysis of H1 in a complex, physiological system may also shed light on the function of H1's globular and tail domains, on functional differences between H1's developmental isoforms, and on the purpose of H1 mitotic phosphorylation, all of which are still unclear.;To address these questions, we have immunodepleted the sole H1 isoform, H1M, from Xenopus egg extracts. Mitotic chromosomes assembled in H1M-depleted extracts are strikingly longer and thinner than wild-type, and fail to cluster properly at the metaphase plate. Addition of recombinant H1M rescues these defects, while H1M overexpression collapses individual chromosomes into an inseparable mass. HIM stabilizes nuclei during decondensation and contributes directly to chromatin structure. Both the conserved, globular domain of H1M and the unstructured carboxyl tail are necessary for efficient chromatin binding and compaction. These experiments establish H1M as a fundamental, rapid compactor of chromatin and reveal a critical role for H1 during chromosome condensation at mitosis.;Developmental isoforms of histone H1 are evolutionarily conserved but poorly understood. When somatic isoforms H1A or H10 are substituted for HIM, they cause chromatin compaction during S-phase, then dissociate from chromosomes during M-phase and fail to rescue chromosome architecture. Nuclear import factors RanBP7 and importin beta bind specifically to somatic H1, but not HIM. Addition of excess RanGTP releases somatic H1 from importins and significantly restores its function on mitotic chromosomes. These behaviors are independent of mitotic Cdk1 phosphorylation, which is unique to somatic H1. Analysis of H1M/H10 chimeras suggests that determinants of chromatin affinity are distributed throughout histone H1. These results reveal distinct regulatory mechanisms among linker histone isoforms, and suggest a specific role for HIM during egg meiotic arrest and rapid embryonic divisions.;To complement the egg extract, we are studying the function and dynamics of H1 isoforms in somatic cells during embryogenesis. After the mid-blastula transition in Xenopus embryos, MA is expressed first in peripheral cells, while H1M is progressively restricted to vegetal cells in a pattern reminiscent of primordial germ cells. H1A stains mitotic chromosomes, suggesting that it may be regulated differently in somatic cells than in egg extracts. Morpholino knockdown of H1A yields underdeveloped embryos that die at tadpole stage, while overexpression of H1A or HIM in results in gastrula cytotoxicity and neural tube closure defects. Functional comparison of H1 isoforms and mutants during development will be bolstered by dynamics studies using H1-GFP fusion proteins, which recover quickly on chromatin after photobleaching. Overall, this body of work identifies the contribution of proper H1 levels to in vivo chromosome architecture as well as vertebrate development, and reveals novel functional differences between embryonic and somatic histone H1 isoforms.