Histone H3 tail positioning and acetylation by the c-Myb but not the v-Myb DNA-binding SANT domain

Abstract

The c-Myb transcription factor coordinates proliferation and differentiation of hematopoietic precursor cells. Myb has three consecutive N-terminal SANT-type repeat domains (R1, R2, R3), two of which (R2, R3) form the DNA-binding domain (DBD). Three amino acid substitutions in R2 alter the way Myb regulates genes and determine the leukemogenicity of the retrovirally transduced v-Myb oncogene. The molecular mechanism of how these mutations unleash the leukemogenic potential of Myb is unknown. Here we demonstrate that the c-Myb-DBD binds to the N-terminal histone tails of H3 and H3.3. C-Myb binding facilitates histone tail acetylation, which is mandatory during activation of prevalent differentiation genes in conjunction with CCAAT enhancer-binding proteins (C/EBP). Leukemogenic mutations in v-Myb eliminate the interaction with H3 and acetylation of H3 tails and abolish activation of endogenous differentiation genes. In primary v-myb-transformed myeloblasts, pharmacologic enhancement of H3 acetylation restored activation of differentiation genes and induced cell differentiation. Our data link a novel chromatin function of c-Myb with lineage-specific expression of differentiation genes and relate the loss of this function with the leukemic conversion of Myb.

Figure 1.

C-Myb-DBD Interacts with the histone H3 tail. (A) Matrix-bound GST-c-Myb-DBD was subjected to purified, recombinant human histones as indicated on the top. Beads were extensively washed, and bound protein was subjected to SDS-PAGE and revealed by silver staining. (B) GST-histone H3.3 fragments (amino acids 1-42 and 42-136), as indicated, or GST control was incubated with in vitro ³⁵S-methionine-labeled c-Myb-DBD. Specifically bound proteins were subjected to SDS-PAGE and revealed by fluorography. (C) Affinity binding as in B using GST-histone tail (human) constructs of H2A, (amino acids 1-35), H2B (amino acids 1-35), H3 (amino acids 1-46), H4 (amino acids 1-34), or H3.3 (amino acids 1-42). (D) Comparison of c-Myb binding to various GST-histone H3 tail constructs. Bottom panels (GST protein) in B-D show Coomassie stain of GST constructs used in the binding experiments as shown above. Input represents 15% of the material used for pull-down assays.

Figure 2.

Configuration of the Myb-DBD and H3 tail binding. (A) Overview of Myb constructs and summary of H3 binding data as shown in B and C. (B) In vitro-translated, ³⁵S-methionine-labeled c-Myb (N- and C-terminally truncated, amino acids 80-497) or v-Myb was incubated with matrix-bound GST-histone tails or GST as a control. (Upper panels) Specifically bound proteins were revealed by SDS-PAGE and fluorography. GST panel shows unspecific binding to GST control, and input panel represents 15% of the labeled material used for pull-down assays. Bottom panel shows protein stains of GST-H3 and GST as indicated. (C) Pull-down of various ³⁵S-methionine-labeled Myb-DBD proteins by GST-H3. Matrix-bound GST fusion proteins were incubated with in vitro-translated, labeled Myb-DBD mutants. Input represents 15% of the labeled material used for pull-down assays.

Figure 3.

Target gene binding of Myb is independent of H3 tail binding. (A) Schematic representations of mim-1 and lysozyme genes indicating the location of amplification products (gray bars), TATA boxes, transcription start sites (+1), and transcription stop sites. (B) Truncated c-Myb proteins (amino acids 80-497), amino acid substitution mutants of c-Myb, or vector controls were transfected into QT6 fibroblasts. Cell lysates were analyzed by ChIP with anti-Myb antibody, and coimmunoprecipitated DNA was PCR amplified, separated by electrophoresis, and shown as a negative image of an ethidium bromide-stained gel. Input lanes (lower panel) represent 0.1% of material used for IP. Myb constructs as shown in Figure 2A. (C) Same as in B using v-Myb and mutants of v-Myb.

Figure 4.

c-Myb and v-Myb proteins recruit p300 to chromatin-embedded promoters independently of H3 tail binding. (A) C-Myb and v-Myb proteins or mutants of c-Myb or v-Myb were transfected together with HA-tagged p300 into QT6 fibroblasts. Chromatin was immunoprecipitated with anti-HA antibody. Coprecipitated DNA was analyzed by PCR amplification. A negative image of an ethidium bromide-stained gel is shown. (Lower panel) Input lanes represent 0.1% of material used for IP. (B) Myb and p300 expression and IP controls. v-Myb proteins were expressed together with p300 in QT6 fibroblasts. (Left) IP with HA or preimmune serum samples were separated by SDS-PAGE, blotted, and revealed by anti-Myb immunostaining. Cell lysate controls of SDS-PAGE separated and blotted proteins show expression of transfected Myb and p300 proteins.

Figure 5.

Acetylation of H3 K18 and K23 at Myb target genes depend on H3 tail binding and p300 recruitment by Myb. (A) Vector control or c-Myb proteins and mutants thereof were transfected into QT6 cells without (left) or together with (right) p300, as indicated on the top. Chromatin was immunoprecipitated with antibodies directed toward acetylated H3 K18 or K23 as shown on the left. Coprecipitated DNA was analyzed by PCR amplification. A negative image of an ethidium bromide-stained gel is shown. Input lanes represent 0.1% of material used for IP. (B) Bone marrow cells were isolated from a 1-d-old chicken and infected with recombinant viruses encoding truncated c-myb or myb-DBD mutants as shown on the right (retroviral constructs indicate lanes of ChIP analysis) and in Introna et al. (1990). Transformed myeloblasts were expanded under appropriate conditions, and ChIP analysis was performed from as indicated on the right.

Figure 6.

The Myb-DBD determines H3 tail acetylation and gene activation in chimeric C/EBPβ-Myb domain swap proteins. (A) Schematic representation of the constructs. C/EBPβ has an N-terminal SWI/SNF recruiting and transactivation domain (βN, amino acids 1-116), a central regulatory domain (RD) and a C-terminal b-Zip domain. The Myb-DBD is indicated as a black box. R2 and R3 Myb repeats are shown as triangles (white and gray triangles represent c-Myb and v-Myb DBD, respectively), and v-Myb mutations are indicated as black dots. The C-terminal transactivating fragment of c-Myb or v-Myb (TAD) are depicted as white and gray boxes. Chimeric constructs consist of the C/EBPβ N terminus (βN) fused in frame to Myb. Chimeric Myb proteins consisted of c-Myb (βN-C-C) or v-Myb (βN-V-V), or domain swaps (βN-C-V; βN-V-C) as indicated. (B, upper panel) Fusion proteins as indicated were translated in vitro in the presence of ³⁵S-methionine and mixed with or without (+/-) in vitro-translated hemagglutinine HA-tagged hBrm (without radioactive label), as indicated. Complexes were immunoprecipitated with a HA-specific antibody (αHA-IP, indicated on the top) and revealed by SDS-PAGE and fluorography. Input lanes represent 10% of material used for IP. (Lower panel) Cell lysate controls of SDS-PAGE separated and blotted proteins to show expression of chimeric proteins. (C) Expression vectors encoding chimeric proteins as indicated were transfected together with the coactivators or combination thereof as indicated on the top. ChIP was performed using antibodies to HA, p300, or acetylated H3 as indicated on the left. Coprecipitated DNA encompassing the mim-1 promoter was analyzed by PCR amplification. A negative image of an ethidium bromide-stained gel is shown. Input lanes represent 0.1% of material used for IP. (D) Expression vectors encoding chimeric proteins as indicated in A or controls (c-myb + C/EBPβ, vector, c-myb, C/EBPβ, v-myb) were transfected into QT6 fibroblasts. RNA was harvested after 24 h, and RNA blots were probed with mim-1 and subsequently with GAPDH as internal control.

Figure 7.

TSA overrides the v-myb differentiation block and activates histone acetylation and myeloid differentiation genes. (A) AMV-transformed myeloblasts were treated without or with TSA (+/- TSA) as indicated. Polyadenylated RNA was analyzed by Northern blotting, and cellular protein was analyzed by SDS-PAGE and Western blotting. (B) Micrographs of AMV-transformed myeloblasts. AMV-myeloblasts grow as suspension cells (-TSA) and acquire an adherent macrophage phenotype when treated with TSA (+TSA) for 48 h.