The linker histone H1 is a highly prevalent protein that compacts chromatin and regulates DNA accessibility and transcription. However, the mechanisms behind H1 regulation of transcription factor (TF) binding within nucleosomes are not well understood. Using in vitro fluorescence assays, we positioned fluorophores throughout human H1 and the nucleosome, then monitored the distance changes between H1 and the histone octamer, H1 and nucleosomal DNA, or nucleosomal DNA and the histone octamer to monitor the H1 movement during TF binding. We found that H1 remains bound to the nucleosome dyad, while the C terminal domain (CTD) releases the linker DNA during nucleosome partial unwrapping and TF binding. In addition, mutational studies revealed that a small 16 amino acid region at the beginning of the H1 CTD is largely responsible for altering nucleosome wrapping and regulating TF binding within nucleosomes. We then investigated physiologically relevant post-translational modifications (PTMs) in human H1 by preparing fully synthetic H1 using convergent hybrid phase native chemical ligation. Both individual PTMs and combinations of phosphorylation and citrullination of H1 had no detectable influence on nucleosome binding and nucleosome wrapping, and had only a minor impact on H1 regulation of TF occupancy within nucleosomes. This suggests that these H1 PTMs function by other mechanisms. Our results highlight the importance of the H1 CTD, in particular, the first 16 amino acids, in regulating nucleosome linker DNA dynamics and TF binding within the nucleosome.

Dynamic chromatin organization instantly influences DNA accessibility through modulating local macromolecular density and interactions, driving changes in transcription activities. Chromatin dynamics has been reported to be locally confined but contributes to the coherent chromatin motion across the entire nucleus. However, the regulation of dynamics, nuclear orientation, and compaction of sub-regions along a single chromosome are not well-understood. We used CRISPR-based real-time single-particle tracking and polymer models to characterize the dynamics of specific genomic loci and determine compaction levels of large human chromosomal domains. Our studies showed that chromosome compaction changed during interphase and the compactions of two arms on chromosome 19 were different. The dynamics of genomic loci were subdiffusive and depended on regions of the chromosome and transcription states. Surprisingly, the correlation between locus nuclear localization and mobility was negligible. Strong tethering interactions detected at the pericentromeric region implies local condensation or associations with organelles at local nuclear microenvironments, such as chromatin-nuclear body association. Based on our findings, we propose a “Guided radial model” for the nuclear orientation of the chromosome 19 long arm.

Nuclear chromosome compaction is non-random and dynamic. The spatial distance among genomic elements instantly modulates transcription. Visualization of the genome organization in the cell nucleus is essential to understand nuclear function. In addition to cell type-dependent organization, high-resolution 3D imaging shows heterogeneous compaction of chromatin organization among the same cell type. Questions remain to be answered if these structural variations were the snapshots of dynamic organization at different time points and if they are functionally different. Live-cell imaging has provided unique insights into dynamic genome organization at short (milliseconds) and long (hours) time scales. The recent development of CRISPR-based imaging opened windows for studying dynamic chromatin organization in single cells in real time. Here we highlight these CRISPR-based imaging techniques and discuss their advances and challenges as a powerful live-cell imaging method that poses high potential to generate paradigm-shifting discoveries and reveal functional implications of dynamic chromatin organization.

H1 is a lysine rich histone that binds nucleosomes and alters nucleosome/chromatin dynamics. Chromatin dynamics, such as chromatin compaction and nucleosome unwrapping, are integral for regulation of fundamental cellular processes including transcription, DNA replication, and DNA repair. Nucleosomal DNA in the entry/exit region spontaneously dissociates from the histone octamer allowing transcription factors (TFs) to bind to DNA in the region when the nucleosome is in an unwrapped state. Instead of blocking wrapping, H1 shifts this equilibrium towards a wrapped state which represses TF binding without fully occluding it. Details of this TF binding mechanism remain unknown, including the structural dynamics of H1 domains during TF binding and which domains affect wrapping and TF binding. Using in vitro fluorescence‐based assays, we have determined the structural dynamics of H1 during TF binding to a nucleosome. H1 binds nucleosomes via electrostatic interactions with DNA and is composed of 3 domains: The winged helix domain that binds the nucleosome dyad and linker DNA, and the intrinsically disordered C terminal and N terminal domains that bind linker DNA. Previous work implied that H1 remains bound to a nucleosome during TF binding, but the details of H1 domain locations were unexplored. We have attached fluorophores and fluorophore quenchers to all 3 H1 domains and either the DNA or histone octamer to measure FRET and fluorophore quenching. This allows us to monitor the movement of the H1 domains relative to the DNA and histone octamer during TF binding. Our results indicate that H1 remains bound to the nucleosome dyad during a TF binding event, and the H1 C terminal domain dissociates from the linker DNA to remain at the dyad. These data further understanding of the mechanistic details of H1 suppression of TF binding to nucleosomes. Future experiments will focus on which H1 domains affect DNA wrapping and TF binding to a nucleosome to further understanding of how H1 regulates TF binding and the implications for transcription. Support or Funding Information NIH R01‐GM121966NIH T32‐GM086252OSU University Fellowship

Cardiac muscle contraction is regulated via Ca2+ exchange with the hetero-trimeric troponin complex located on the thin filament. Binding of Ca2+ to cardiac troponin C, a Ca2+ sensing subunit within the troponin complex, results in a series of conformational re-arrangements among the thin filament components, leading to an increase in the formation of actomyosin cross-bridges and muscle contraction. Ultimately, a decline in intracellular Ca2+ leads to the dissociation of Ca2+ from troponin C, inhibiting cross-bridge cycling and initiating muscle relaxation. Therefore, troponin C plays a crucial role in the regulation of cardiac muscle contraction and relaxation. Naturally occurring and engineered mutations in troponin C can lead to altered interactions among components of the thin filament and to aberrant Ca2+ binding and exchange with the thin filament. Mutations in troponin C have been associated with various forms of cardiac disease, including hypertrophic, restrictive, dilated, and left ventricular noncompaction cardiomyopathies. Despite progress made to date, more information from human studies, biophysical characterizations, and animal models is required for a clearer understanding of disease drivers that lead to cardiomyopathies. The unique use of engineered cardiac troponin C with the L48Q mutation that had been thoroughly characterized and genetically introduced into mouse myocardium clearly demonstrates that Ca2+ sensitization in and of itself should not necessarily be considered a disease driver. This opens the door for small molecule and protein engineering strategies to help boost impaired systolic function. On the other hand, the engineered troponin C mutants (I61Q and D73N), genetically introduced into mouse myocardium, demonstrate that Ca2+ desensitization under basal conditions may be a driving factor for dilated cardiomyopathy. In addition to enhancing our knowledge of molecular mechanisms that trigger hypertrophy, dilation, morbidity, and mortality, these cardiomyopathy mouse models could be used to test novel treatment strategies for cardiovascular diseases. In this review, we will discuss (1) the various ways mutations in cardiac troponin C might lead to disease; (2) relevant data on mutations in cardiac troponin C linked to human disease, and (3) all currently existing mouse models containing cardiac troponin C mutations (disease-associated and engineered).