H3K4 mono- and di-methyltransferase MLL4 is required for enhancer activation during cell differentiation
- National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, United States
- The George Washington University, United States
- Korea Basic Science Institute, Republic of Korea
- National Institute of Arthritis, Musculoskeletal, and Skin Diseases, National Institutes of Health, United States
Figures
Figure 1 with 2 supplements
MLL4 is required for brown adipose tissue and muscle development.
(A and B) Generation of Mll4 conditional KO mice (Mll4f/f). (A) Schematic representation of mouse Mll4 wild-type (WT) allele, targeted allele, conditional KO (flox) allele and KO allele. In the …
Figure 1—figure supplement 1
Generation of Mll3 and Mll4 whole body KO mice.
(A) Protein domains in yeast SET1 and the homologous mouse SET1-like H3K4 methyltransferases. (B–E) Generation of Mll3 and Mll4 whole body KO mice. Wild-type (WT) and KO alleles of Mll3 and Mll4 …
Figure 1—figure supplement 2
Confirmation of Mll4 deletion.
Immortalized Mll3−/−Mll4f/f brown preadipocytes were infected with adenoviral GFP or Cre. (A) Genome browser view of RNA-Seq analysis on Mll4 gene locus. The targeted exons 16–19 are highlighted in …
Figure 2 with 2 supplements
MLL4 controls induction of cell-type-specific genes during differentiation.
(A–E) Adipogenesis of Mll3−/−Mll4f/f brown preadipocytes. (A and B) MLL4 is required for adipogenesis. Immortalized Mll3−/−Mll4f/f brown preadipocytes were infected with adenoviral GFP or Cre, …
Figure 2—figure supplement 1
MLL4 is required for adipogenesis.
(A–C) MLL3 is dispensable for adipogenesis of brown preadipocytes. Brown preadipocytes isolated from Mll3+/+ and Mll3−/− E18.5 embryos were immortalized by retroviruses expressing SV40T. Cells were …
Figure 2—figure supplement 2
Confirmation of RNA-Seq data by qRT-PCR.
https://doi.org/10.7554/eLife.01503.008
Figure 3 with 2 supplements
Cell-type- and differentiation-stage-specific genomic binding of MLL4.
Adipogenesis and myogenesis were done as in Figure 2A,F,G, respectively. Cells were collected for ChIP-Seq analysis of MLL4. (A) Venn diagram of MLL4 binding regions at day 0 (preadipocytes), day 2 …
Figure 3—figure supplement 1
MLL4 binds to adipogenesis genes.
ChIP-Seq of MLL4 was performed during adipogenesis as described in Figure 2. (A–E) MLL4 binding on adipogenic genes at day 2 of adipogenesis. In each panel, the top 2 tracks show the raw data while …
Figure 3—figure supplement 2
ChIP-qPCR confirmation of ChIP-Seq data.
(A) Schematic of genomic locations of MLL4+ enhancers on Pparg, Cebpa, Fabp4 (aP2) and Prdm16 loci. E, enhancer. (B) ChIP-qPCR confirmation of ChIP-Seq data on MLL4+ enhancers.
Figure 4 with 3 supplements
Genomic co-localization of MLL4 with lineage-determining TFs during differentiation.
(A) Adipogenic TF binding motifs are enriched at MLL4 binding regions during adipogenesis while myogenic TF binding motifs are enriched at MLL4 binding regions in myocytes. Top 2,000 emergent MLL4 …
Figure 4—figure supplement 1
ChIP-Seq and RNA-Seq data on Pparg gene during adipogenesis.
D0 and D2, day 0 and day 2.
Figure 4—figure supplement 2
ChIP-Seq and RNA-Seq data on Cebpa gene during adipogenesis.
https://doi.org/10.7554/eLife.01503.014
Figure 4—figure supplement 3
PPARγ interacts with MLL3/MLL4-containing H3K4 methyltransferase complex in cells.
(A) Anti-FLAG M2 agarose was used to immunoprecipitate from nuclear extracts of HeLaS cells stably expressing FLAG-tagged PPARγ (F-PPARγ) (Ge et al., 2008). The immunoprecipitates were analyzed by …
Figure 5 with 1 supplement
MLL4 co-localizes with lineage-determining TFs on active enhancers during differentiation.
ChIP-Seq analyses of MLL4, TFs, H3K4me1/2/3 and H3K27ac were done at day 2 of adipogenesis. (A) Table depicting histone modifications used to define gene regulatory elements. TSS, transcription …
Figure 5—figure supplement 1
MLL4 co-localizes with lineage-determining TFs on active enhancers during myogenesis.
MyoD-stimulated myogenesis of brown preadipocytes was done as in Figure 2. (A) Average binding profiles of MLL4 and MyoD around the center of each type of gene regulatory elements in myocytes. (B) …
Figure 6 with 1 supplement
MLL4 is a major H3K4 mono- and di-methyltransferase in cells.
(A and B) In vitro histone methyltransferase (HMT) assay. (A) SET1A/SET1B complex (SET1A/B.com) and MLL3/MLL4 complex (MLL3/4.com) that were affinity-purified from 293T nuclear extracts were …
Figure 6—figure supplement 1
MLL3 and MLL4 are H3K4 mono- and di-methyltransferases in mammalian cells.
(A) Western blot analyses of histone modifications in Mll3−/− brown preadipocytes. (B) Western blot analyses of histone modifications before and after MyoD-stimulated myogenesis. Pre-ad, …
Figure 7 with 2 supplements
MLL4 is required for enhancer activation during differentiation.
Mll3−/−Mll4f/f brown preadipocytes were infected with adenoviral GFP or Cre as in Figure 2. Cells were collected at day 0 and day 2 of adipogenesis for ChIP-Seq of H3K4me1/2/3, H3K27ac, MED1 and Pol …
Figure 7—figure supplement 1
MLL4 is required for H3K4me1/2 on MLL4+ promoters.
The average profiles of H3K4me1/2/3 levels on the 480 (29 silent plus 451 active) MLL4+ promoters identified during adipogenesis (Figure 5C) are shown.
Figure 7—figure supplement 2
MLL4 is required for enhancer activation during myogenesis.
MyoD-stimulated myogenesis of brown preadipocytes was done as in Figure 2. (A) Deletion of MLL4 markedly decreases H3K4me1 and H3K27ac levels on MLL4+ MyoD+ active enhancers. Average profiles of …
Figure 8 with 1 supplement
C/EBPβ recruits and requires MLL4 to establish a subset of adipogenic enhancers.
Mll3−/−Mll4f/f brown preadipocytes were infected with retroviral C/EBPβ or Vec only, and then infected with adenoviral GFP or Cre. Cells were collected 2 days after confluence without induction of …
Figure 8—figure supplement 1
Time-course ChIP-qPCR on C/EBPβ+MLL4+ enhancers on Pparg gene locus.
(A) Schematic of C/EBPβ+MLL4+ adipogenic enhancers 1, 2, and 3 (E1, E2 and E3) on Pparg gene locus. (B) Time-course ChIP-qPCR on C/EBPβ+MLL4+ enhancers on Pparg gene locus at 0, 2, 4, 8, 12, 24, and …
Additional files
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Supplementary file 1
(A) Mapped read counts in RNA-Seq and ChIP-Seq. (B) List of SYBR Green primers for ChIP-qPCR
- https://doi.org/10.7554/eLife.01503.025
