Quaas CE et al. (2022), Transcription suppression is mediated by the HD...

XB-ART-59398

J Biol Chem 2022 Nov 01;29811:102578. doi: 10.1016/j.jbc.2022.102578.

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Transcription suppression is mediated by the HDAC1-Sin3 complex in Xenopus nucleoplasmic extract.

Quaas CE , Lin B , Long DT .

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Modification of histones provides a dynamic mechanism to regulate chromatin structure and access to DNA. Histone acetylation, in particular, plays a prominent role in controlling the interaction between DNA, histones, and other chromatin-associated proteins. Defects in histone acetylation patterns interfere with normal gene expression and underlie a wide range of human diseases. Here, we utilize Xenopus egg extracts to investigate how changes in histone acetylation influence transcription of a defined gene construct. We show that inhibition of histone deacetylase 1 and 2 (HDAC1/2) specifically counteracts transcription suppression by preventing chromatin compaction and deacetylation of histone residues H4K5 and H4K8. Acetylation of these sites supports binding of the chromatin reader and transcription regulator BRD4. We also identify HDAC1 as the primary driver of transcription suppression and show that this activity is mediated through the Sin3 histone deacetylase complex. These findings highlight functional differences between HDAC1 and HDAC2, which are often considered to be functionally redundant, and provide additional molecular context for their activity.

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Species referenced: Xenopus laevis
Genes referenced: actb brd4 hdac1 hdac2 hdac3 hdac8 isyna1 mta2 rps3a sin3a
GO keywords: histone acetylation [+]

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	Figure 1. Transcribed plasmids become inactivated during incubation in extract. A, schematic of pActin gene construct. B, pActin was incubated in NPE supplemented with buffer or an RNase inhibitor (RNasin). RNA was isolated and quantified by RT-qPCR at the times indicated (n = 2). Validation of RNasin activity is shown in Fig. S1. C, pActin was incubated in NPE supplemented with excess rNTPs or ARM. RNA was isolated and quantified after 120 min (n = 2). D, about 5 to 25 ng/μl pActin was incubated in NPE. RNA was isolated and quantified at the times indicated (n = 2). E, about 10 ng/μl pActin was incubated in NPE for 60 min 0 to 20 ng/μl of additional pActin was then added, and RNA was isolated and quantified after 120 min (n = 2). Student t test: p-value < 0.05 (∗), p-value < 0.01 (∗∗), p-value < 0.001 (∗∗∗), not significant (n.s.). Error bars represent ±1 SD. NPE, nucleoplasmic extract; RT-qPCR, reverse transcription quantitative PCR.
	Figure 2. Inhibition of HDAC1/2 counteracts transcription suppression and increases DNA accessibility. A, pActin was incubated in NPE supplemented with 100 μM of the indicated HDAC inhibitor. RNA was isolated and quantified after 120 min (n = 3). B, pActin and pActinΔTATA were incubated in NPE supplemented with 10 ng/μl α-amanitin and/or 100 μM Romidepsin, as indicated. RNA was isolated and quantified after 120 min (n = 2). C, pActin was incubated in NPE supplemented with buffer or 100 μM Romidepsin. RNA was isolated and quantified at the indicated time points (n = 2). D, pActin was incubated in NPE for 90 min. Reactions were then supplemented with either buffer or 100 μM Romidepsin. RNA was isolated and quantified at the indicated time points (n = 2). E, about 30 ng/μl pActin was incubated in NPE supplemented with buffer or Romidepsin for 120 min. Samples were then analyzed by the MNase cleavage assay (n = 2). Supercoiled (SC). F, quantification of SC plasmid intensity in (E) (n = 2). G, sperm chromatin was incubated in NPE supplemented with buffer or 100 μM Romidepsin. At the indicated time points, samples were withdrawn and visualized by phase contrast light microscopy (n = 2). H, quantification of two-dimensional chromatin area from (G). Student t test: p-value < 0.05 (∗), p-value < 0.01 (∗∗), p-value < 0.001 (∗∗∗), not significant (n.s.). Error bars represent ±1 SD. NPE, nucleoplasmic extract.
	Figure 3. Romidepsin stimulates BRD4-dependent transcription. A, plasmid pull-down schematic. B, pActin was incubated in NPE supplemented with buffer or 100 μM Romidepsin. At the indicated time points, DNA-bound proteins were isolated by plasmid pull down and visualized by Western blot with the indicated antibodies (n = 2). Input represents 3% of total reaction sample. Acetylation of H4K5 and H4K8 in total reaction samples is shown in Fig. S3A. C, pActin was incubated in NPE supplemented with buffer or 100 μM Romidepsin. Samples were withdrawn at the indicated time points and binding of BRD4 to the actb promoter region was analyzed by ChIP (n = 3). D, pActin was incubated in NPE supplemented with Romidepsin and/or JQ1, as indicated. RNA was isolated and quantified after 120 min (n = 2). Student t test: p-value < 0.05 (∗), p-value < 0.01 (∗∗), p-value < 0.001 (∗∗∗), not significant (n.s.). Error bars represent ±1 SD. BRD, bromodomain; NPE, nucleoplasmic extract.

	Figure 5. The Sin3 deacetylase complex promotes transcription suppression. A, sequential IP schematic. B, mock, HDAC1, or HDAC2 IPs were performed in NPE. The supernatants from HDAC1 or HDAC2 IPs were then used for a second round of IPs using the converse antibody. Isolated proteins were then analyzed by Western blot with the indicated antibodies (n = 3). 10% of total reaction sample (IN), supernatant (S), pellet (P). C, NPE was immunodepleted using preimmune (ΔMock) or MTA2 (ΔMTA2) antibodies. Depleted extracts were analyzed by Western blot using the indicated antibodies. D, pActin was incubated in mock- or MTA2-depleted extract. RNA was isolated and quantified after 120 min (n = 2). E, NPE was immunodepleted using preimmune (ΔMock) or Sin3a (ΔSin3a) antibodies. Depleted extracts were analyzed by Western blot using the indicated antibodies. F, pActin was incubated in mock- or Sin3a-depleted extract. RNA was isolated and quantified after 120 min (n = 2). G, NPE was immunodepleted using preimmune (ΔMock) or HDAC1 (ΔHDAC1) antibodies. HDAC1-depleted extract was then supplemented with immunoprecipitated proteins recovered by preimmune (+Mock IP) or Sin3a (+Sin3a IP) IP. pActin was incubated in each extract and RNA was isolated and quantified after 120 min (n = 2). Student t test: p-value < 0.05 (∗), p-value < 0.01 (∗∗), p-value < 0.001 (∗∗∗), not significant (n.s.). Error bars represent ±1 SD. IP, immunoprecipitation; NPE, nucleoplasmic extract.
	Figure S1. RNA degradation and protection. To validate the use of RNasin in extract, NPE was supplemented with buffer, RNase, or a combination of RNase and RNasin and incubated at RT for 20 minutes. Samples were then resolved by agarose gel electrophoresis and the presence of endogenous RNA was visualized with SYBR Gold stain (n=2).
	Figure S2. (A) pActin was incubated in NPE supplemented with buffer or the indicated amount of SAHA, Romidepsin, RGFP966, or Santacruzamate A. RNA was isolated and quantified after 120 minutes (n=3). Error bars represent +/- one standard deviation. (B-C) pCMV (B) or pBRCA1 (C) was incubated in NPE supplemented with buffer or 100 µM Romidepsin. RNA was isolated and quantified after 120 minutes (n=2). Student t-test: p-value < 0.05 (), p-value < 0.01 (), p-value < 0.001 (**), not significant (n.s.). Error bars represent +/- one standard deviation.
	Figure S3. Total histone acetylation and undetected modifications. (A) Total reaction samples from Figure 3B were analyzed by Western blot with the indicated antibodies (n=2). (B) pActin was incubated in NPE supplemented with buffer or 100 µM Romidepsin. At the indicated time points, DNA-bound proteins were isolated by plasmid pull-down and visualized by Western blot with the indicated antibodies (n=3). Input represents 3% of total reaction sample. Note that lack of signal may represent low histone modification or low activity by the indicated antibodies. (C) pActin was incubated in NPE supplemented with buffer or Romidepsin for 60 minutes. Samples were withdrawn for IP with pre-immune (Mock) or RNAPII antibodies and isolated proteins were analyzed by Western blot with the indicated antibodies (n=2).
	Figure S4. Characterization of Xenopus laevis HDAC1 and HDAC2 antibodies. (A) NPE was immunodepleted using pre-immune (∆Mock), HDAC1 (∆HDAC1), or HDAC2 (∆HDAC2) antibodies. Different amounts of depleted extract were then loaded to analyze the extent of HDAC1 and HDAC2 depletion in each extract by Western blot (n=3).
	Figure S5. MTA2 depletion reduces BRD4 binding. (A) NPE was immunodepleted using pre-immune (∆Mock) or MTA2 (∆MTA2) antibodies. Depleted extracts were analyzed by Western blot using the indicated antibodies (n=2). MTA2 blot is reproduced from Figure 5C for comparison. (B) pActin was incubated in mock- or MTA2-depleted extract. Samples were withdrawn after 90 minutes and binding of BRD4 to the actb promoter region was analyzed by ChIP (n=2). Student t-test: p-value < 0.05 (), p-value < 0.01 (), p-value < 0.001 (**), not significant (n.s.). Error bars represent +/- one standard deviation.
	Figure 1. Transcribed plasmids become inactivated during incubation in extract.A, schematic of pActin gene construct. B, pActin was incubated in NPE supplemented with buffer or an RNase inhibitor (RNasin). RNA was isolated and quantified by RT-qPCR at the times indicated (n = 2). Validation of RNasin activity is shown in Fig. S1. C, pActin was incubated in NPE supplemented with excess rNTPs or ARM. RNA was isolated and quantified after 120 min (n = 2). D, about 5 to 25 ng/μl pActin was incubated in NPE. RNA was isolated and quantified at the times indicated (n = 2). E, about 10 ng/μl pActin was incubated in NPE for 60 min 0 to 20 ng/μl of additional pActin was then added, and RNA was isolated and quantified after 120 min (n = 2). Student t test: p-value < 0.05 (∗), p-value < 0.01 (∗∗), p-value < 0.001 (∗∗∗), not significant (n.s.). Error bars represent ±1 SD. NPE, nucleoplasmic extract; RT-qPCR, reverse transcription quantitative PCR.
	Figure 2. Inhibition of HDAC1/2 counteracts transcription suppression and increases DNA accessibility.A, pActin was incubated in NPE supplemented with 100 μM of the indicated HDAC inhibitor. RNA was isolated and quantified after 120 min (n = 3). B, pActin and pActinΔTATA were incubated in NPE supplemented with 10 ng/μl α-amanitin and/or 100 μM Romidepsin, as indicated. RNA was isolated and quantified after 120 min (n = 2). C, pActin was incubated in NPE supplemented with buffer or 100 μM Romidepsin. RNA was isolated and quantified at the indicated time points (n = 2). D, pActin was incubated in NPE for 90 min. Reactions were then supplemented with either buffer or 100 μM Romidepsin. RNA was isolated and quantified at the indicated time points (n = 2). E, about 30 ng/μl pActin was incubated in NPE supplemented with buffer or Romidepsin for 120 min. Samples were then analyzed by the MNase cleavage assay (n = 2). Supercoiled (SC). F, quantification of SC plasmid intensity in (E) (n = 2). G, sperm chromatin was incubated in NPE supplemented with buffer or 100 μM Romidepsin. At the indicated time points, samples were withdrawn and visualized by phase contrast light microscopy (n = 2). H, quantification of two-dimensional chromatin area from (G). Student t test: p-value < 0.05 (∗), p-value < 0.01 (∗∗), p-value < 0.001 (∗∗∗), not significant (n.s.). Error bars represent ±1 SD. NPE, nucleoplasmic extract.
	Figure 3. Romidepsin stimulates BRD4-dependent transcription.A, plasmid pull-down schematic. B, pActin was incubated in NPE supplemented with buffer or 100 μM Romidepsin. At the indicated time points, DNA-bound proteins were isolated by plasmid pull down and visualized by Western blot with the indicated antibodies (n = 2). Input represents 3% of total reaction sample. Acetylation of H4K5 and H4K8 in total reaction samples is shown in Fig. S3A. C, pActin was incubated in NPE supplemented with buffer or 100 μM Romidepsin. Samples were withdrawn at the indicated time points and binding of BRD4 to the actb promoter region was analyzed by ChIP (n = 3). D, pActin was incubated in NPE supplemented with Romidepsin and/or JQ1, as indicated. RNA was isolated and quantified after 120 min (n = 2). Student t test: p-value < 0.05 (∗), p-value < 0.01 (∗∗), p-value < 0.001 (∗∗∗), not significant (n.s.). Error bars represent ±1 SD. BRD, bromodomain; NPE, nucleoplasmic extract.
	Figure 4. HDAC1 drives transcription suppression.A, schematic representation of HDAC1 and HDAC2. Unique amino acid sequences used to generate peptide antigens are shown at the C terminus for each protein. B, pActin was incubated in NPE supplemented with buffer or Romidepsin. Samples were withdrawn at the indicated time points and binding of HDAC1 and HDAC2 to the actb promoter region was analyzed by ChIP (n = 3). Significance shown for comparison between +Buffer and +Romidepsin reactions. C, NPE was immunodepleted using preimmune (ΔMock), HDAC1 (ΔHDAC1), HDAC2 (ΔHDAC2), or a combination HDAC1 and 2 (ΔHDAC1&2) antibodies. Depleted extracts were analyzed by Western blot with the indicated antibodies. D, pActin was incubated in depleted extracts from (C). RNA was isolated and quantified after 120 min (n = 3). E, pActin was incubated in mock-, HDAC1-, or HDAC2-depleted extract for 120 min. Samples were then analyzed by the MNase cleavage assay (n = 2). Supercoiled (SC). F, quantification of SC plasmid intensity in (E). G, sperm chromatin was incubated in mock-, HDAC1-, or HDAC2-depleted extracts. At the indicated time points, samples were withdrawn and visualized by phase contrast light microscopy (n = 2). H, quantification of two-dimensional chromatin area in (G). Student t test: p-value < 0.05 (∗), p-value < 0.01 (∗∗), p-value < 0.001 (∗∗∗), not significant (n.s.). Error bars represent ±1 SD. ChIP, chromatin immunoprecipitation; NPE, nucleoplasmic extract.
	Figure 5. The Sin3 deacetylase complex promotes transcription suppression.A, sequential IP schematic. B, mock, HDAC1, or HDAC2 IPs were performed in NPE. The supernatants from HDAC1 or HDAC2 IPs were then used for a second round of IPs using the converse antibody. Isolated proteins were then analyzed by Western blot with the indicated antibodies (n = 3). 10% of total reaction sample (IN), supernatant (S), pellet (P). C, NPE was immunodepleted using preimmune (ΔMock) or MTA2 (ΔMTA2) antibodies. Depleted extracts were analyzed by Western blot using the indicated antibodies. D, pActin was incubated in mock- or MTA2-depleted extract. RNA was isolated and quantified after 120 min (n = 2). E, NPE was immunodepleted using preimmune (ΔMock) or Sin3a (ΔSin3a) antibodies. Depleted extracts were analyzed by Western blot using the indicated antibodies. F, pActin was incubated in mock- or Sin3a-depleted extract. RNA was isolated and quantified after 120 min (n = 2). G, NPE was immunodepleted using preimmune (ΔMock) or HDAC1 (ΔHDAC1) antibodies. HDAC1-depleted extract was then supplemented with immunoprecipitated proteins recovered by preimmune (+Mock IP) or Sin3a (+Sin3a IP) IP. pActin was incubated in each extract and RNA was isolated and quantified after 120 min (n = 2). Student t test: p-value < 0.05 (∗), p-value < 0.01 (∗∗), p-value < 0.001 (∗∗∗), not significant (n.s.). Error bars represent ±1 SD. IP, immunoprecipitation; NPE, nucleoplasmic extract.

References [+] :

Allis, The molecular hallmarks of epigenetic control. 2016, Pubmed