Regulation of transcription by the MLL2 complex and MLL complex-associated AKAP95

Transcription

1 Supplementary Information Regulation of transcription by the complex and MLL complex-associated Hao Jiang, Xiangdong Lu, Miho Shimada, Yali Dou, Zhanyun Tang, and Robert G. Roeder

2 Input HeLa NE IP lot: p53 SAM oa -N RNA - ASH2L WDR5 Menin ND.6 p3 D H4 WT WT Mut WT WT Mut Mut Mut acetyl- K4me acetyl-h4 K4me3 H4 K4me Supplementary Figure. complex and its activities in K4 methylation and in chromatintemplated transcription. (A) Immunoblots for proteins immunoprecipitated from nuclear extract derived from HeLa cells by indicated antibodies. () Immunoblots for in vitro histone acetylation assay with histone octamer substrates. () Autoradiographic analysis of in vitro transcription on a chromatin template with additions of complex, p3, SAM and acetyl-oa as indicated. The relative transcription levels are shown in the bar chart below. (D) Immunoblots for in vitro HMT assay on chromatin assembled with histone octamers containing wild type or (K4R K8R) mutant and wild type H4 or H4 (K5R K8R) mutant by the complex.

3 f293 NE IP: FH-DPY3 NE IP: HA WDR5 FP Input M2 Transfect: FH-ASH2L HA lot: long exposure short exposure Input Amylose Transfect: ASH2L MP -MP -MP MP degradation products lot: MP ASH2L WDR5 Supplementary Figure 2. is associated with MLL complexes. (A) Immunoblot analysis of FH-DPY3-MLL complexes following immunoprecipitation (IP) by anti- HA or normal mouse IgG antibodies of nuclear extracts from FH-DPY3 and control (f293) cell lines. () Immunoblot analysis for anti-flag M2 agarose-mediated immunoprecipitation for 293T cells transfected with with or without Flag-HA-ASH2L (FH-ASH2L). () Immunoblot analysis for amylose resin-mediated precipitation for 293T cells transfected with Maltose inding Protein (MP) or -MP as indicated, all together with ASH2L expression construct.

4 o-activation fold Luc assay Vector MLL Relative mrna levels RT-PR Vector MLL o-activation fold ng ng HA GAPDH Supplementary Figure 3. Overexpression of, but not certain point mutants, enhances luciferase gene expression (A) Luciferase assay for G-293T cells overexpressing MLL or in the presence of Gal4- VP6. The luciferase signal from cells transfected with empty vector was arbitrarily set at, and the error bars represent the range of values from two independent transfections. () Quantitative RT-PR results for luciferase mrna levels in G-293T cells overexpressing MLL or in the presence of Gal4-VP6. The error bars indicate s.d. from triplicate measurements from one representative experiment (out of two experiments). () Luciferase assay (above) and immunoblot analysis (below) for G-293T cells transfected with 25 or ng of plasmids encoding WT or indicated point mutants in the presence of Gal4- VP6. The luciferase signal from cells transfected with empty vector (together with Gal4-VP6) was arbitrarily set at, and the error bars represent the range of values from two independent transfections.

5 hromatin p hromatin Supplementary Figure 4. enhances -mediated K4 methylation on chromatin. Autoradiographic analysis of in vitro HMT on chromatin by the complex in the presence of [ 3 H]- SAM. : coomassie blue staining.

6 shrna: Scramble Relative cell numbers shrna: Scramble Days of culture Relative mrna levels shrna: Scramble Nanog Oct4 Klf4 Sox2 D Percent Input shrna: Scramble sh # sh #2 Supplementary Figure 5. Self-renewal of mouse ESs is not affected by depletion of (A) Morphology of E4 cell clones days after infection with Scramble or shrna #2. () Proliferation assay days after infection with Scramble shrna or shrna #2, and the error bars indicate s.d. from three independent seedings. () RT-qPR results for expression of genes involved in ES maintenance. In () and (D), the error bars indicate s.d. from triplicate measurements from one representative experiment (out of two experiments). (D) DPY3 hip on control and mouse ESs stably depleted of by two different shrnas, followed by qpr on a subset of gene promoters from previous studies.

7 DPY3 KD KD (P =.66e -57 ) DPY3 KD RbP5 KD DPY3 KD RbP5 KD KD Supplementary Figure 6. Partial overlap of genes affected on their post-atra expression levels by, DPY3 and RbP5 knockdown (KD). Venn diagram analyses of genes whose post-atra expression levels were reduced over.5 fold by depletion of indicated proteins in E4 cells. P value was calculated by Fisher s exact test.

8 DPY3 KD KD DNA binding Transcription regulation Neuron differentiation Developmental protein Neuron differentiation Proteinaceous extracellular matrix Extracellular matrix Developmental protein Pattern specification process Embryonic skeletal system development Vasculature development Anterior/posterior pattern formation Pattern specification process Extracellular matrix Signal Anterior/posterior pattern formation DNA binding Transcription regulation E E-4 E-8 E-2 P value E-6 E-2 E E-4 E-8 E-2 P value E-6 E-2 DPY3 KD and RbP5 KD D DPY3 KD and KD DNA binding Developmental protein Transcription regulation Anterior/posterior pattern formation Developmental protein Neuron differentiation Neuron differentiation Proteinaceous extracellular matrix Extracellular matrix Pattern specification process Anterior/posterior pattern formation Embryonic skeletal system development DNA binding Pattern specification process Extracellular matrix Transcription regulation Embryonic skeletal system development Signal E E-4 E-8 E-2 P value E-6 E-2 E E-4 E-8 E-2 P value E-6 E-2 E DPY3 KD, RbP5 KD, and KD Developmental protein Pattern specification process Neuron differentiation Anterior/posterior pattern formation Proteinaceous extracellular matrix Extracellular matrix Skeletal system development DNA binding Transcription regulation Signal Supplementary Figure 7. ommon and differential enrichment of pathways for genes affected at the post-atra expression levels by, DPY3 and RbP5 knockdown (KD). Gene ontology analyses of genes whose post-atra expression levels were commonly reduced over.5 fold by indicated individual knockdowns (in the chart titles) in mouse ESs. E E-4 E-8 E-2 E-6 E-2 P value

9 p53 SAM and oa DNA p53 p3 PR2 SAM and oa hromatin RNA HeLa NE IP: Input Transfection of Flag-HA-WDR5: Flag IP 5 95 WDR WDR HA (WDR5)

10 D 2 M2 M2-FH- DPY3 Input E Supplementary Figure 8. Uncropped key figures. (A) Related to Fig. 2. () Related to Fig. 3. () Related to Fig. 3. (D) Related to Fig. 5F. (E) Related to Fig. 6A.

11 Supplementary Table. sirna and shrna sequences RNA format Species target Sequence 5-3 # GGAAAGAAGTAGAATT shrna Mouse #2 GTGTTTGTATGAAGTTT ON-TARGET plus SMARTpool sirna Human GUAUGGUGAUA GGAAGAGUGAAUAGU GAAAAAGAAUAUAGUG GAUGAGGUGUAUG Supplementary Table 2. Primers App. Species gene Forward start* Forward primer (5-3 ) ackward start* ackward primer (5-3 ) RT mouse GAPDH ATTTTTGTGAGTGAG GGAAAATTATTTG RT mouse Nanog TAGAGATGAAGAAT TTAAATGGTTTTTTG RT mouse Oct4 GTATGGGGTTT GGGAGTTAAATGTT RT mouse Klf4 GTTTTTAATTTGTTGATTTGGG AGATGTAGATG RT mouse Sox2 GGGAAAAAAGAGT TGGGAAGGTGTAT RT mouse IGFP5 TTTAGGAGTTAAAG GGTAAGTTGGGAGGTAA RT mouse Msx TTTGGGAAGAAAAAG GGGTATTTTGAAGA RT mouse Hox6 TGGATGAGGAATGAAT GAGTTAGGTAGGGTTGAA RT Luciferase AATAAATATTGGATATGG AAATTATAATTAAGAGATG RT human GAPDH TTATTGATAATAATGG TGTTGGAAGATGGTGATGGG RT human HAND GAGTGATAAGT GTAGGGAGAGTTTGATT RT human Msx AAGTTGAGAAGAGTA TTTATGGTTAGTGA hip Gal4- Luciferase hip mouse Intergenic hr8: 72,86, TTATGGTATGTAATGAGTAA AAGGGGTTGTTAAAAA hr8: 72,86,24 GGGGATATGGTTGTGA AGAGTATGGAGGTAGA hip mouse Actin 42 AGGATATAGAAGGAAAT 234 TAAGTAGGGTAAAGTTGGT hip mouse IGFP5 89 TGATTTAAATTTT 257 TGGAGAAAGGTAAGAG hip mouse IGFP5 downstream 399 GATGTGGGTTTG downstream 466 GTGTGGGTTATGATTTGA hip mouse Hox6 38 GAGGAAGAAAG 45 ATAATGTGGAATTAT hip mouse IGFP5 89 TGATTTAAATTTT 257 TGGAGAAAGGTAAGAG hip mouse Msx 26 GGTGTGTATGATTTTTG 3 GGAGTTATTTGAA hip mouse GAPDH 236 TTGGGAGTAATTATTT 32 AATAGTTAAGATT *Starting positions for the forward and backward primers relative to the gene TSS. Supplemental Reference. Jiang, H. et al. Role for Dpy-3 in ES ell-fate Specification by Regulation of K4 Methylation within ivalent Domains. ell 44, (2).