Preferential Phosphorylation on Old Histones during Early Mitosis in Human Cells

Abstract

How histone post-translational modifications (PTMs) are inherited through the cell cycle remains poorly understood. Canonical histones are made in the S phase of the cell cycle. Combining mass spectrometry-based technologies and stable isotope labeling by amino acids in cell culture, we question the distribution of multiple histone PTMs on old versus new histones in synchronized human cells. We show that histone PTMs can be grouped into three categories according to their distributions. Most lysine mono-methylation and acetylation PTMs are either symmetrically distributed on old and new histones or are enriched on new histones. In contrast, most di-and tri-methylation PTMs are enriched on old histones, suggesting that the inheritance of different PTMs is regulated distinctly. Intriguingly, old and new histones are distinct in their phosphorylation status during early mitosis in the following three human cell types: HeLa, 293T, and human foreskin fibroblast cells. The mitotic hallmark H3S10ph is predominantly associated with old H3 at early mitosis and becomes symmetric with the progression of mitosis. This same distribution was observed with other mitotic phosphorylation marks, including H3T3/T6ph, H3.1/ 2S28ph, and H1.4S26ph but not S28/S31ph on the H3 variant H3.3. Although H3S10ph often associates with the neighboring Lys-9 di-or tri-methylations, they are not required for the asymmetric distribution of Ser-10 phosphorylation on the same H3 tail. Inhibition of the kinase Aurora B does not change the distribution despite significant reduction of H3S10ph levels. However, K9me2 abundance on the new H3 is significantly reduced after Aurora B inhibition, suggesting a cross-talk between H3S10ph and H3K9me2.In eukaryotes, histone proteins facilitate the packaging of DNA molecules. The DNA double helix wraps around histone octamers to form nucleosomes. A histone octamer contains two copies of each core histone H3, H4, H2A, and H2B. A 5th histone, histone H1, is associated with the linker DNA which lies between the nucleosomes. Canonical histone proteins are cell cycle-dependent and are produced in S phase (1, 2), whereas cell cycle-independent histone variants (e.g. H3.3) are synthesized throughout the cell cycle (3). Histone proteins carry numerous post-translational modifications (PTMs) 3 that are involved in multiple functions such as epigenetic regulation of transcription, DNA damage repair, and cell cycle progression (4, 5). To maintain lineage identity and to guide proper transcription, cells must replicate PTMs from old histones onto new histones at each cell division. Major efforts have been devoted to understanding how histones themselves are transmitted through the DNA replication fork in S phase (6). In principle, the newly deposited nucleosomes could contain entirely old or newly synthesized histone proteins, or a mixture of both. Accumulating evidence suggests that most H3/H4 tetramers remain intact, with the exception of some H3.3/H4 tetramers, indicating that nucleosomes should contain either new or ...

Symmetry from Asymmetry or Asymmetry from Symmetry?

“…Mitotic hallmarks, such as H3S10P, H3T3/T6P, H3.1/2S28P, and H1.4S26P, are shown to be predominantly associated with old histones at early mitosis in cultured human cell lines (Lin et al, 2016). We wanted to define the mechanism(s) by which sister chromatids might be recognized and segregated in an asymmetric manner.…”

Epigenetic characteristics of the mitotic chromosome in 1D and 3D

“…Although new nucleosomes are incorporated directly after DNA duplication, the histone modifications are added in the time between S-phase up till early G1. A recent study by Alabert et al showed that not all histone modifications are added to the new nucleosomes at the same time (Alabert et al, 2015; Lin, Yuan, Han, Marchione, & Garcia, 2016). …”

The Inherent Asymmetry of DNA Replication

“…New histones are incorporated as necessary to maintain nucleosome density. As new histones are largely devoid of PTMs (Lin et al, 2016), old histones must play an instructive role by assisting new histones in the process of acquiring the appropriate epigenetic signatures needed to recapitulate the parental chromatin state. In support of this hypothesis, chromatin maturation studies have demonstrated that for a variety of PTMs, old histones are able to recruit the appropriate histone modifying enzymes needed to established the correct PTMs on newly synthesized histones (Alabert et al, 2015, Alabert and Groth, 2012, Ayyanathan et al, 2003, Hansen et al, 2008, Margueron et al, 2009, Ragunathan et al, 2015, Audergon et al, 2015).…”

“…Although whether centromeric regions are replicated in such a coordinated fashion has yet to be demonstrated, investigations have revealed that epigenetic asymmetries underlie the proper recognition and segregation of sister chromatids bearing distinct asymmetric signatures during ACD of the Drosophila GSC (Figure 13). The team of Xie et al was recently able to demonstrate that a peri-centromere-enriched, mitosis-specific phosphorylation of threonine three in H3 (H3T3P) selectively labels old histones during the transition from prophase to metaphase (Xie et al, 2015, Lin et al, 2016). Furthermore, the authors found that this asymmetry serves as a mechanism to allow the mitotic spindle to recognize sister chromatids enriched with different populations of histones as a means to assure their proper segregation during GSC ACD.…”