The molecular characteristics of chromatin states and the histone modifying enzymes catalysing histone PTMs have been studied extensively and a picture emerges in which a complex network of factors function to maintain chromatin homeostasis or remodel chromatin to confer proper genome function. Nonetheless, the temporal dynamics of chromatin and interplay between
chromatin factors and factors involved in other key cellular processes are just now starting to be unravelled. An analysis of heterochromatin dynamics at the mating-type region of the fission yeast Schizosaccharomyces pombe is presented here. Heterochromatin establishment experiments were performed in which the sole H3K9me methyltransferase Clr4 was re-introduced through a genetic cross into clr4Δ cells and gene silencing was measured at the single-cell level by fluorescent reporter genes integrated at strategic positions in the mating-type region. These experiments have revealed important functions of cis-acting factors in heterochromatin establishment, namely the inverted repeats flanking the mating-type region and the two binding sites for the transcription factor Atf1. In addition, it was found that the establishment rate depended greatly on the size of the mating-type region, with larger domains establishing more slowly. Integrating these experimental results with mathematical modelling provided a model for the dynamics during heterochromatin establishment in which the ability of histone deacetylases to act globally within the heterochromatic system is a key feature. This may be facilitated by a specific 3-dimensional conformation conferred by the inverted repeats and by action of the HP1 homolog Swi6.
Genetic screens have revealed the requirements for many factors involved in DNA replication to be important for heterochromatic silencing. The replisome component Mrc1 was the main focus of investigation into these factors where role in silencing had been assigned to maintaining heterochromatin rather than establishing. The function had been mapped to the HBS domain, however despite the previously described the role of the HBS domain in recruiting the DDK kinase Hsk1 and in regulation of origin firing this was found not to be important for the role of Mrc1.
More detailed analysis of the HBS domain revealed the requirement of conserved residues in maintaining silencing which includes an acidic patch. Analysis the CMG helicase subunit Mcm2 showing histone chaperone activity indicated a similar pattern of loss-of-silencing and epistasis analysis indicated that they two factors may work through a common pathway. Furthermore, mimicking histone chaperone activity by tethering the FACT subunit Pob3 to Mrc1ΔHBS partially rescued silencing defects. This shines light on how replisome components could work together to maintain heterochromatic domains by facilitating inheritance of parental histones during DNA replication.