Histone proteomics implicates H3K36me2 and its regulators in mouse embryonic stem cell pluripotency exit and lineage choice

mESC growth and differentiation

mESCs were cultured in serum-free conditions prior to neuroectoderm differentiation as previously described [9]. mESCs were differentiated into neuroectoderm following a monolayer adherent differentiation protocol [10] with minor modifications as previously described [11]. For RNA isolation, 0.5 × 10⁶ cells were collected and stored in TRIzol™ (15596018; Thermo Fisher Scientific) at −20 °C. Endoderm differentiation was performed as previously described [11]. 2 × 10⁶ cells were collected from ESC state and from the fifth day of endoderm/neuroectoderm differentiation for histone extraction.

Histone acid extraction

Total histone proteins were isolated by acid extraction method as described [12]. Protein concentration was measured with Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific).

Histone proteomics

Cells stored as in 2.1 were sent to the Broad Institute Proteomics Platform. Histones were extracted and proteomic analysis was performed as described [13, 14]. Results were normalized to total histone in the sample and to the unique normalization peptide of where the modification is found (H3K4me0 peptide for normalization of H3 modifications, for example) [13]. The results are provided as Supplementary File and visualized in GraphPad Prism (Supplemental Figure 1).

Figure 1:

Quantitative analysis of histone 3 modifications during mESC differentiation. Proteomic analysis of H3 peptides in mESCs and (A) and (C) neuroectoderm and (B) and (D) endoderm committed wild-type cells. The abundance of each modified peptide was normalized to H3K4me0 peptide in that particular cell type. The results of two biological replicates are shown.

Western blot

Western blot was performed as previously described [15]. Antibodies used: H3K36me2 (Abcam, ab176921), H3K36me3 (CST, d5a7) H3 (Santa Cruz, sc-517576). The band intensities were quantified using ImageJ software and normalized to H3.

RNA isolation, cDNA conversion and RT-qPCR

Total RNA was isolated by using Qiagen RNeasy Plus Micro Kit (Qiagen, Valencia, CA, USA) following the provided protocol. 500 ng RNA was converted to cDNA using iScript™ cDNA Synthesis Kit (Bio-Rad Laboratories, Hercules, CA, USA). Information about primer sequences can be found in Table 1.

RT-qPCR was performed with SsoAdvanced™ Universal SYBR^® Green Supermix (Bio-Rad Laboratories, Inc., Hercules, California, USA) using BioRad CFX Connect™ Real-Time PCR Detection System. Expression levels were normalized to β-actin.

Statistical analyses

Data normality and variance homogeneity were assessed via Shapiro-Wilk and Levene’s tests, respectively, in R. GraphPad’s Prism 9 was used for graph generation and conducting multiple unpaired Student’s t-tests. Spearman correlation test was performed in R to evaluate the relationship between H3K36me2 levels (quantified by Western blot) and the expression of selected genes on mESC state and fifth day of neuroectoderm differentiation. Correlation coefficients and p-values were shown in Table 2.

H3K36 dimethylation levels are increased during early stages of neuroectoderm differentiation

The aim was to infer which chromatin factors play a role for the mESC exit from pluripotency and the choice between endoderm and neuroectoderm lineages. To that end, histone modification changes were quantitatively catalogued through proteomic analysis of histone proteins in wild-type mESCs and cells differentiated towards endoderm or neuroectoderm lineages. The purpose of this method is to correctly identify the chemical modifications and the amino-acids they are linked to in histones. Histone proteomics is technically challenging due to their small size and the abundance of lysines and arginines which are generally used for cleavage reactions prior to mass spectrometry. Chemical modifications at these residues might alter cleavage efficiency or might be labile to sample preparation protocol. To ensure success, the analysis was performed in the proteomics facility of the Broad Institute that specializes in histone proteomics.

As mESCs differentiate, they lose the activity of the pluripotency network that keep them at the stem cell state and start expressing lineage specific TFs dependent upon intra- and extracellular signals. These pioneer TFs then start a cascade of cell character change through activation of several downstream TFs and eventually lead to differential molecular organization and more mature cell types. The earliest emergence of lineage specific TFs was focused on, with endoderm-committed cells collected when Foxa2 TF is first expressed, and neuroectoderm-committed cells collected when Sox1 is first expressed. These markers were identified by others as the first transcription factors expressed and led the differentiation towards said lineage and have been successfully used to study mESC early differentiation [10, 14, 16], [17], [18], [19], [20]. Monitoring for the earliest signs of lineage specific marker expression helped focus at a tight window of change, from pluripotent state to lineage commitment. This approach can highlight the earlier and more direct histone modification alterations that guide lineage choice.

The abundance of histone peptides in each sample was first normalized to total histone level and then individually to the relevant histone level. Since the growth conditions of mESCs differ for endoderm or neuroectoderm commitment, mESC samples were collected independently for these conditions and the data for differentiated cells were normalized to their respective mESC samples.

No significant alterations in H2A or H2B modification levels were observed upon endoderm or neuroectoderm differentiation (Supplemental Figure 1A and B). Acetylated H4 levels decreased upon mESC differentiation (Supplemental Figure 1C and D), consistent with a role of histone deacetylases at pluripotency exit [14]. This study is focused on H3 modifications since they showed a larger overall change upon differentiation and some offered lineage-specific effects (Figure 1A–D).

H3S10 phosphorylation, a marker for active mitosis [21], was abundantly found in ESC state compared to differentiated states (Figure 1C and D), consistent with rapid cycling of mESCs. Elevated H3K9 and H3K36 methylation levels were observed in a neuroectoderm differentiation specific manner (Figure 1A, C). Considering the wider range of difference observed for H3K36 methylation, this modification was selected as the focus of this study. H3K36 dimethylation (H3K36me2) increase was more pronounced than H3K36 trimethylation (H3K36me3) and the profile was not affected by the presence or absence of H3K27 acetylation or monomethylation (Figure 1A). To validate these findings with Western blot, mESCs grown in serum-free conditions were differentiated towards neuroectoderm lineage [11]. Morphological changes associated with differentiation were observed (Figure 2A), where the round and three-dimensional mESC colonies gradually flattened and assumed epithelial-like morphology. The success of differentiation was verified with RT-qPCR analysis of key pluripotency markers (Nanog, Pou5f1 (Oct4); Figure 2B) and neuroectoderm lineage specific genes (Sox1, Pax6, N-cadherin; Figure 2C). Consistent with previous literature [19], the earliest neural lineage marker Sox1 level peaked at the third day of neuroectoderm differentiation while its downstream targets Pax6 and N-cadherin levels showed a progressive increase through day 5 of differentiation, confirming functional outcome of SOX1 expression. These trends were accompanied by a sharp decrease in the expression of both pluripotency markers; Nanog and Pou5f1. Western blot analyses of histone extracts confirmed that H3K36me2 level is substantially elevated on the fifth day of neuroectoderm differentiation compared to mESC state while H3K36me3 level remained unchanged (Figure 2D). However, both H3K36me2 and H3K36me3 levels remain fairly similar during endoderm differentiation (Figure 2E), suggesting a unique role of H3K36me2 during neuroectoderm lineage choice rather than at pluripotency exit.

Figure 2:

Validation of mass spectrometry results in mESCs and differentiation (A) Light microscopy images of wild-type mESCs at mESC stage and during neuroectoderm differentiation time-course. Cell images were obtained using Olympus CKX-53 microscope at 100× magnification in native medium. Expression levels of (B) pluripotency and (C) neuroectoderm lineage markers during neuroectoderm differentiation time-course measured by RT-qPCR analysis. Results are normalized to β-actin levels. The error bars represent SEM of at least three biological replicates. X-axis shows days of neuroectoderm differentiation. Day 0 is mESC stage. Western blot analyses of H3K36me2 and H3K36me3 using histone extracts from mESCs or (D) neuroectoderm and (E) endoderm directed cells (Day 5). Histone 3 (H3) was used as loading control. The results are representative of at least two biological replicates. The intensities of H3K36me2 and H3K36me3 were quantified using ImageJ software and normalized to H3. Relative intensities of H3K36me2 and H3K36me3 are shown in bar graphs as fold over H3, where error bars represent SEM of at least two biological replicates. Statistical analyses (Student’s t-test) were done by using GraphPad Prism software. *: p-value<0.05. Nonsignificant values are not shown.

Transcriptional levels of H3K36 methyltransferases during the course of neuroectoderm differentiation

Level of H3K36 dimethylation is regulated by methyltransferases and demethylases through the addition of two methyl groups to H3K36, or through the demethylation of H3K36me3 [6], respectively. First, the expression levels of methyltransferases that add up to two methyl groups to H3K36 were investigated; namely Nsd1-3, Setd3, Ash1l, Setmar and Smyd2.

RT-qPCR analyses through neuroectoderm differentiation time-course revealed a significant decrease in Nsd1 expression level (Figure 3A) and an increase in Nsd2 level (Figure 3B). A strong negative correlation was found between Nsd1 and H3K36me2 level and a significant positive correlation was observed between Nsd2 and H3K36me2 (Table 2). Nsd3 level was stable through neuroectoderm differentiation (Figure 3C). No significant trends were observed in the expression levels of Ash1l, Setmar and Smyd2 through neuroectoderm differentiation time course (Figure 3D–F).

Figure 3:

Expression level of H3K36 methyltransferases. Expression levels of histone methyltransferases for H3K36 during neuroectoderm differentiation time-course measured by RT-qPCR analysis. Results are normalized to β-actin levels. The error bars represent SEM of at least three biological replicates. Statistical analysis (Student’s t-test) was done using GraphPad Prism software. *: p-value<0.05; **: p-value<0.01. Nonsignificant values are not shown.

We next focused on trimethyltransferases (Setd2 and Setd5) for a possible decrease in their levels which may lead to accumulated H3K36me2 [5, 22]. Although no pronounced trend was observed in Setd2 expression (Figure 3G), Setd5 displayed a downward trend (Figure 3H) during neuroectoderm differentiation. Since H3K36me2 is the substrate for trimethyltransferases, a decrease in Setd5 might be associated with the H3K36me2 accumulation during neuroectoderm differentiation (Table 2). Expression of another SET domain containing protein, Setd3, showed a less pronounced downward trend (Figure 3I).

Transcriptional levels of H3K36 demethylases during the course of neuroectoderm differentiation

In order to obtain a more comprehensive perspective, H3K36 demethylases were investigated next. H3K36 demethylation was reported to have crucial roles for mESC self-renewal [23]. KDM4B (JMJD2B) and KDM4C (JMJD2C) have cell and context dependent functions and act as H3K36me2/3 and H3K9me2/3 demethylases. KDM2A and KDM2B are demethylases specific to H3K36me1/2 [24].

Although no significant trend in Kdm2a,b and Kdm4a,b levels was found during the course of neuroectoderm differentiation (Supplemental Figure 2A–D), Kdm4c level decreased through neuroectoderm commitment (Supplemental Figure 2E). This decrease might be associated with decreased H3K36me2 demethylation activity which might explain the accumulation of H3K36me2 observed in neuroectoderm differentiation (Table 2).

Transcriptional levels of H3K36me2/3 readers during the course of neuroectoderm differentiation

Histone methylations exert their function through reader proteins that can specifically recognize the methylated histones and recruit downstream proteins [3].

The expression levels of H3K36me3 readers PHF1 (PCL1) and PHF19 (PCL3) were quite low in mESC state and during neuroectoderm differentiation (data not shown) [4]. DNMT3A and DNMT3B are de novo DNA methyltransferases that establish the DNA methylation patterns during early embryonic development [25]. They contain H3K36me2/3 recognition PWWP domains [26]. A prominent decrease was observed in Dnmt3a (Supplemental Figure 3A) while Dnmt3b expression seemed to peak on the third day of differentiation (Supplemental Figure 3B).

H3K36me2/3 reader MSH6 is a part of DNA mismatch repair (MMR) machinery [27]. Msh6 expression has declined through neuroectoderm differentiation (Supplemental Figure 3C) which negatively correlated with H3K36me2 level change (Table 2).

ZMYND11 is involved in regulation of transcriptional elongation and mRNA splicing. It specifically recognizes H3.3K36me3 variant [8], enriched in actively transcribed regions. A progressive increase observed in Zmynd11 (Supplemental Figure 3C) implicates a possible role in neuroectoderm differentiation.

Some degree of increase in Psip1 (Supplemental Figure 3E) and decrease in Mrg15 (Supplemental Figure 3F) were observed, though statistically insignificant.

Transcriptional levels of selected genes during the course of endoderm differentiation

The RT-qPCR analyses revealed expression level changes of genes related to H3K36 methylation during early neuroectoderm lineage commitment. To identify whether these changes are unique for neuroectoderm lineage or rather they are associated with the exit of pluripotency, we investigated the expression of these genes during endoderm differentiation using transcriptomics data (unpublished data). Visualization of RNA-seq data through IGV shows a gradual decrease in pluripotency TFs Oct4, Sox2, Nanog and Klf4 (Supplemental Figure 4A). As the earliest marker of definitive endoderm [16], Foxa2 is expressed at day 3 of differentiation, along with meso/endoderm lineage TF Gsc (Supplemental Figure 4B). Downstream targets of FOXA2 (Sox17, Gata4, Gata6 and Hnf4a) emerge at differentiation day 4 (Supplemental Figure 4B), confirming functional outcome of Foxa2 expression.

IGV (Supplemental Figure 4C) and differential expression (Supplemental Figure 4D) analyses showed that similar to neuroectoderm, Nsd2 and Zmynd11 expression increases significantly in endoderm lineage. Kdm4c and Msh6 expression levels decreased significantly in endoderm as they did in neuroectoderm lineage. However, the decrease in Nsd1 level is unique for neuroectoderm differentiation (Figure 3A) with no corresponding significant change in endoderm differentiation (Supplemental Figure 4C and D). Dnmt3b level increased and remained high during the course of endoderm differentiation (Supplemental Figure 4C and D). Thus, this protein may have functions at different time points for endoderm and neuroectoderm differentiation. While Setd5 was trending downward during neuroectoderm differentiation (Figure 3H), an upward trend was observed in endoderm differentiation (Supplemental Figure 4C). Since H3K36me2 is used as a substrate for trimethylation by SETD5, such a unique decrease encountered in neuroectoderm differentiation time course might explain the accumulation of H3K36me2.