A reconfigured pattern of MLL occupancy within mitotic chromatin promotes rapid transcriptional reactivation following mitotic exit

Abstract

Mixed lineage leukemia (MLL) and its metazoan Trithorax orthologs have been linked with the epigenetic maintenance of transcriptional activity. To identify mechanisms by which MLL perpetuates active transcription in dividing cells, we investigated its role during M phase of the cell cycle. Unlike other chromatin-modifying enzymes examined, we found that MLL associates with gene promoters packaged within condensed mitotic chromosomes. Genome-wide location analysis identified a globally rearranged pattern of MLL occupancy during mitosis in a manner favoring genes that were highly transcribed during interphase. Knockdown experiments revealed that MLL retention at gene promoters during mitosis accelerates transcription reactivation following mitotic exit. MLL tethers Menin, RbBP5, and ASH2L to its occupied sites during mitosis, but is dispensable for preserving histone H3K4 methylation. These findings implicate mitotic bookmarking as a component of Trithorax-based gene regulation, which may facilitate inheritance of active gene expression states during cell division.

Figure 1

MLL N- and C- fragments localize globally with mitotic chromatin. Immunofluorescence (IF) of asynchronous HeLa cells stained with A) anti-MLL-C (468), B) anti-MLL-N (457), C) anti-SETD1A, D) anti-RNA polymerase II (N20), or E) anti-LSD1 (09–058) antibodies. All secondary antibodies were conjugated with Cy2 and counterstained with DAPI.

Figure 2

ChIP-chip analysis of MLL occupancy in interphase and mitosis. A, B, C) Scaled log₂-ratio data for the MEIS1, HOXC11, and EEF1A1 promoter regions. Green horizontal bars denote regions called as peaks. D) Venn diagram evaluating the relative frequency of MLL occupancy at overlapping and unique peak locations between interphase and mitosis. E) Percentile rank of mRNA abundance for genes bound by MLL or Pol II. Vertical line represents the percentage of genes expected to be bound if no relationship existed between occupancy and expression. Chi-squared statistical test was used. * indicates p < 0.05. ** indicates p < 10⁻¹⁰. *** indicates p < 10⁻²⁰

Figure 3

MLL occupancy is acquired at several highly expressed genes upon mitotic silencing. ChIP-qPCR analysis using the indicated antibodies comparing interphase and mitotic preparations of chromatin. The anti-MLL-C (468) or anti-MLL-N (456) antibody was used. (AG) Primer-pairs amplify at +0.5 kb relative to the TSS. (H) Primers amplify indicated location relative to the PABPC1 TSS. Interphase (blue) refers to cells arrested by double-thymidine block. Mitosis (red) refers to cells arrested by sequential thymidine-nocodazole treatment. CD4 is a constitutively silent control gene. Error bars denote S.E.M.

Figure 4

MLL is required for rapid post-mitotic reactivation of its occupied genes. A) Western blot of whole-cell extracts prepared from cells expressing control or MLL-specific shRNA. B) ChIP q-PCR with anti-MLL-N (456) antibodies performed in mitotic cells. C-J) Primary transcript RT-qPCR analysis of the indicated genes in cells released from mitotic arrest at the indicated timepoints. Signals were normalized to the level of stable GAPDH mature transcript. Asterisks indicate p < 0.05 calculated using a two-tailed student’s t-test. Error bars denote S.E.M. K-L) Timecourse ChIP-qPCR performed with the indicated antibodies using primers amplifying the PABPC1 +0.5 kb region.

Figure 5

RbBP5, ASH2L, and Menin occupy gene promoters during mitosis. ChIP-qPCR analysis using the indicated antibodies comparing occupancy between interphase and mitosis, as described in Figure 3.

Figure 6

MLL is required for the association of Menin, RbBP5, and ASH2L with mitotic chromatin. A, B, C, G, H) ChIP-qPCR performed in control or MLL-shRNA expressing HeLa cells arrested in mitosis. D, E) Timecourse ChIP-qPCR performed in cells released from nocodazole arrest at the indicated timepoints. F) Western blot of acid extracted histones prepared from control or MLL-shRNA expressing cells. Error bars denote S.E.M.

Figure 7

MLL-dependent HOX genes display MLL occupancy in both interphase and mitosis. A) RT-qPCR performed in control or MLL-shRNA expressing HeLa cells. B) ChIP-qPCR with anti-MLL-N antibodies (456) using the indicated primers, which amplify at +0.5 kb relative to the transcription start site. C) UCSC genome browser track (Kent et al., 2002) showing the HOXC cluster. qPCR amplicons are denoted by labeled horizontal bars. D-G) ChIP qPCR with the indicated antibodies comparing interphase, mitotic preparations of chromatin in cells expressing either control hairpin (shSCR) or MLL-specific hairpins (shMLL). Error bars denote S.E.M.