Mediator coordinates PIC assembly with recruitment of CHD1

Abstract

Murine Chd1 (chromodomain helicase DNA-binding protein 1), a chromodomain-containing chromatin remodeling protein, is necessary for embryonic stem (ES) cell pluripotency. Chd1 binds to nucleosomes trimethylated at histone 3 Lys 4 (H3K4me3) near the beginning of active genes but not to bivalent domains also containing H3K27me3. To address the mechanism of this specificity, we reproduced H3K4me3- and CHD1-stimulated gene activation in HeLa extracts. Multidimensional protein identification technology (MuDPIT) and immunoblot analyses of purified preinitiation complexes (PICs) revealed the recruitment of CHD1 to naive chromatin but enhancement on H3K4me3 chromatin. Studies in depleted extracts showed that the Mediator coactivator complex, which controls PIC assembly, is also necessary for CHD1 recruitment. MuDPIT analyses of CHD1-associated proteins support the recruitment data and reveal numerous components of the PIC, including Mediator. In vivo, CHD1 and Mediator are recruited to an inducible gene, and genome-wide binding of the two proteins correlates well with active gene transcription in mouse ES cells. Finally, coimmunoprecipitation of CHD1 and Mediator from cell extracts can be ablated by shRNA knockdown of a specific Mediator subunit. Our data support a model in which the Mediator coordinates PIC assembly along with the recruitment of CHD1. The combined action of the PIC and H3K4me3 provides specificity in targeting CHD1 to active genes.

Figure 1.

H3K4me3 stimulates in vitro transcription from nuclear extract. (A) Schematic of the trimethyl-lysine analog synthesis at histone 3 Cys 4. Below the schematic is a Western blot showing the specific detection of H3K4me3 MLA by antibody (Abcam anti-K4me3). (B) Schematic of the immobilized chromatin template and chromatin normalization. The extent of chromatin assembly was monitored by EMSA; equivalent amounts of chromatin were used in all experiments. (C) Schematic and autoradiograph of in vitro transcription as detected by primer extension on unmodified (Naive) and synthetic H3K4me3 chromatin templates in the presence and absence of the activator GAL4-VP16 and acetyl-CoA (AcCoA) using HeLa nuclear extract. (AcCoA was necessary for optimal levels of transcription.) (D) Signal quantitation and statistical analysis of the stimulation by H3K4me3. Three repeats of the transcription experiment were quantitated using Imagequant TL software, graphed, and subjected to a Student's t-test as a measure of statistical significance between naive unmodified and H3K4me3 chromatin.

Figure 2.

H3K4me3 effectors are recruited to the PIC. (A) MuDPIT analyses were performed on PICs assembled on chromatin arrays: a chart of PIC components, H3K4me3 effector proteins, and other factors recruited. Average enrichment in the presence of GAL4-VP16 and further enrichment on H3K4me3 chromatin are shown as a heat map from average NSAFe5 values for the factors in the complex. Average NSAFe5 values for each complex are ranked by percentile in a three-color gradient from high (red) to medium (yellow) to low (blue) as shown. (B) Time courses of PIC recruitment in HeLa extracts were compared between Naive and H3K4me3 templates by immunoblotting to validate enrichment of select factors as determined by the MuDPIT screen. (C) Quantitation and statistical analysis of Western blot signals for CHD1 and Mediator enriched on H3K4me3 versus Naive chromatin. Signals were normalized to VP16. Student's t-test was used to calculate the statistical significance of the differences at the 60-min time point. Assays were performed in triplicate.

Figure 3.

Mediator plays a key role in CHD1 recruitment to the PIC. (A) Immunoblot of Mediator subunits and control proteins in Mediator-depleted (ΔMediator) and mock-depleted (Mock) nuclear extract loaded in threefold steps. (B) Immunoblot (left panel) and quantitation (right panel) comparing CHD1 recruitment time courses in an immobilized template assay (IT) on H3K4me3 chromatin in Mock versus ΔMediator extracts. Western blot signal was quantified using LiCOR imaging software, and the statistical significance between Mock- and Mediator-depleted extracts was calculated using Student's t-test. Signals were normalized to VP16. (C) CHD1 recruitment in Mock- versus Mediator-depleted extracts repeated using naked DNA templates.

Figure 4.

Characterization of CHD1 recruitment using purified complexes. (A) Immunoblot analysis of CHD1 recruitment to naked DNA templates in a Mediator-depleted extract supplemented with highly purified Mediator ± activator. Mediator was normalized to levels found in the HeLa nuclear extract used in Figures 2 and 3. The top panel is a representative immunoblot of the Mediator complementation experiment, while the bottom panels are graphs analyzing triplicate experiments. Quantitation was performed using LiCOR imaging software. Signals were normalized to VP16. (B) Chart of PIC-related complexes found associated with recombinant Flag-tagged human CHD1 incubated with HeLa nuclear extract at higher and lower heparin conditions. Average NSAFe5 values for each complex are ranked by percentile in a three-color gradient from high (red) to mid (yellow) to low (blue) as shown. Note that the cutoff values differ from the chart in Figure 2.

Figure 5.

Characterization of CHD1 during transcription initiation. (A) Autoradiograph of nucleosome remodeling assay demonstrating that recombinant CHD1 is enzymatically active on Naive and H3K4me3 ³²P-labeled 601 mononucleosomal templates. (B) Western blot of CHD1-depleted nuclear extract and a silver-stained gel of purified recombinant Flag-CHD1 expressed in baculovirus. (C) Autoradiograph of in vitro transcription primer extension assays using CHD1-depleted nuclear extracts as shown. Transcription was compared between CHD1-depleted (ΔCHD1) and mock-depleted nuclear extracts and rescued with the addition of purified CHD1 as shown. Signals were quantitated using Imagequant TL and normalized to Mock-treated + activator. (D) Graph showing CHD1 recruitment to H3K4me3 versus Naive immobilized chromatin templates with purified Mediator and activator. Trace levels of CHD1 were detected by immunoblotting in the purified Mediator alone. CHD1 recruitment in each lane was normalized to recruited Mediator. (E) Levels of transcription were measured in a rate-limiting assay performed as illustrated by the schematic. H3K4me3 chromatin templates were preincubated with activator, Mediator, and CHD1 as indicated. After 30 min, unbound protein was removed, and buffer with or without ATP was added. After a further 30-min incubation, the buffer was removed and beads were suspended in transcription buffer containing HeLa nuclear extract and nucleotides. After 10 min, the transcription reaction was subjected to primer extension analysis to measure mRNA. Signals were quantitated using Imagequant TL and normalized to the signal in lane 6.

Figure 6.

Chd1 and Mediator binding correlate in vivo. (A) ChIP analysis of doxycycline-induced enrichment of VP16, Med1-containing Mediator, CHD1, and Pol II on a stably integrated doxycycline-inducible Luciferase reporter in U2OS cells during a time course. (B) Venn diagram showing distribution and overlap of genes for Chd1 and Med1 binding and H3K4me3 (excluding H3K27me3) occupancy across the mouse ES cell genome. P-values are as follows: Chd1-Med1, P-value = 1.42 × 10⁻²¹⁶; Chd1-H3K4me3 (excluding H3K27me3), P-value < 9.34 × 10⁻³²²; Med1-H3K4me3 (excluding H3K27me3), P-value < 1.05 × 10⁻³²¹. (C) Box plot of gene expression levels of Chd1-positive, Med1-positive, and H3K4me3 (excluding H3K27me3)-positive genes and all other genes, including singly and doubly bound as well as unbound genes. Chd1-, Med1-, and H3K4me3-cobound genes show an overall higher level of transcription. For triple-positive versus all other genes: D = 0.4276, P-value < 2.2 × 10⁻¹⁶.

Figure 7.

CHD1 associates with Med1-containing Mediator in vivo. Immunoblots showing specific shRNA knockdown of Med1 and Med23 subunits of Mediator in 293T cells (left panels) and Med1-dependent coimmunoprecipitation of Mediator by targeted immunoprecipitation of CHD1 (right panel). Subunits representing the head (Med6), middle (Med7), and tail (Med14) modules of Mediator are blotted.