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A single N-terminal amino acid determines the distinct roles of histones H3 and H3.3 in the Drosophila male germline stem cell lineage [1]

['Chinmayi Chandrasekhara', 'Department Of Biology', 'The Johns Hopkins University', 'Baltimore', 'Maryland', 'United States Of America', 'Rajesh Ranjan', 'Howard Hughes Medical Institute', 'Jennifer A. Urban', 'Brendon E. M. Davis']

Date: 2023-05

Adult stem cells undergo asymmetric cell divisions to produce 2 daughter cells with distinct cell fates: one capable of self-renewal and the other committed for differentiation. Misregulation of this delicate balance can lead to cancer and tissue degeneration. During asymmetric division of Drosophila male germline stem cells (GSCs), preexisting (old) and newly synthesized histone H3 are differentially segregated, whereas old and new histone variant H3.3 are more equally inherited. However, what underlies these distinct inheritance patterns remains unknown. Here, we report that the N-terminal tails of H3 and H3.3 are critical for their inheritance patterns, as well as GSC maintenance and proper differentiation. H3 and H3.3 differ at the 31st position in their N-termini with Alanine for H3 and Serine for H3.3. By swapping these 2 amino acids, we generated 2 mutant histones (i.e., H3A31S and H3.3S31A). Upon expressing them in the early-stage germline, we identified opposing phenotypes: overpopulation of early-stage germ cells in the H3A31S-expressing testes and significant germ cell loss in testes expressing the H3.3S31A. Asymmetric H3 inheritance is disrupted in the H3A31S-expressing GSCs, due to misincorporation of old histones between sister chromatids during DNA replication. Furthermore, H3.3S31A mutation accelerates old histone turnover in the GSCs. Finally, using a modified Chromatin Immunocleavage assay on early-stage germ cells, we found that H3A31S has enhanced occupancy at promoters and transcription starting sites compared with H3, while H3.3S31A is more enriched at transcriptionally silent intergenic regions compared to H3.3. Overall, these results suggest that the 31st amino acids for both H3 and H3.3 are critical for their proper genomic occupancy and function. Together, our findings indicate a critical role for the different amino acid composition of the N-terminal tails between H3 and H3.3 in an endogenous stem cell lineage and provide insights into the importance of proper histone inheritance in specifying cell fates and regulating cellular differentiation.

Funding: This work was supported by the National Institutes of Health (F32 GM134664 to C.C., K99 GM145973 to J.A.U., 5T32GM007231 to B.D. and J.S., F31 HD104526 to J.S., and R35GM127075 to X.C., as well as Division of Intramural Research, NHLBI to K.Z.), American Cancer Society grant# 133950-PF-19-131-01-DMC to J.A.U, the Howard Hughes Medical Institute, the David and Lucile Packard Foundation, and Johns Hopkins University startup funds to X.C. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.

Recent studies have shown that point mutations at multiple residues of histones and histone variants result in dramatic cellular defects and human diseases [ 36 , 37 ]. Our previous work showed that mutations at the Threonine 3 residue of H3 [e.g., H3T3A (Thr3 to Ala) and H3T3D (Thr3 to Asp)] lead to Drosophila male GSC loss, progenitor germline tumors, and progressively decreased male fertility [ 38 ]. Mutations at critical histone and histone variant residues have also been identified in a variety of human cancers [ 36 ]. For example, mutation at the Lys27 residue of H3.3 [H3.3K27M (Lys27 to Met)] or H3.1 (H3K27M) have both been identified in pediatric glioblastoma [ 2 , 37 , 39 ]. Another critical residue is located at position 31 of H3 and H3.3. This is the only distinct amino acid between H3 and H3.3 at their N-termini ( Fig 1B ), which has been shown to be mutated in human ovarian cancer, squamous cell carcinomas, and colorectal adenocarcinomas samples [ 36 ]. To better examine the importance of this residue, we swapped this particular amino acid in both H3 and H3.3 to generate 2 hybrid mutant histone proteins ( Fig 1B ). We then expressed each of them in the Drosophila early-stage male germline to investigate whether the mutant histones lead to any germline defect. Our data demonstrate a spectrum of phenotypes, indicating that the 31st residue plays a vital role in regulating proper germline activities. Collectively, our studies provide in vivo insights into how the N-terminal protein composition differences between H3 and H3.3 dictate their roles in specifying distinct cell fates and their inheritance patterns during ACD of male GSCs.

Previously, it has been reported that during ACD of Drosophila male GSC, preexisting (old) canonical histones H3 and H4 are selectively inherited by the GSC, whereas newly synthesized (new) H3 and H4 are enriched in the differentiating daughter GB cell [ 29 , 30 ]. Intriguingly, this phenomenon is unique to the canonical histones H3 and H4. By contrast, histone variant H3.3 does not exhibit such a global asymmetric pattern [ 29 ]. While significant progress has been made in understanding how epigenetically distinct sister chromatids enriched with old versus new H3 are specifically established and recognized, our understanding of what underlies the observed differences between H3 and H3.3 inheritance patterns during ACD of GSCs is limited. Histone variants, such as H3.3, H2A.Z, and CENP-A, are involved in important biological functions, such as transcription, DNA repair, and defining centromeres [ 31 – 34 ]. Between H3 and H3.3, H3 is primarily incorporated during DNA replication, while H3.3 is incorporated in a replication-independent manner [ 33 , 35 ].

( A ) An illustration of the apical tip of the Drosophila testis. The hub (cyan) is a cluster of 10–12 densely packed somatic cells. The GSCs (dark green) and the CySCs (magenta) are radially positioned around the hub, with 2 CySCs enveloping each GSC. The GSCs undergo an asymmetric division to produce a self-renewed daughter GSC and a GB (light green). The GB subsequently leaves the hub and undergoes 4 rounds of miotic divisions with incomplete cytokinesis to create cysts of interconnected SGs. The differentiating germline cysts continue to be encapsulated by 2 postmitotic cyst cells (orange). Round spectrosome (purple) is detected in early-stage germline including GSCs and GBs, which is branched as fusome (purple) in late-stage SGs. Adapted from [ 54 ]. ( B ) Diagram of the single amino acid differences at the N-terminal tails in WT H3 and H3.3 proteins. A point mutation in the N-terminal tail of H3 changing alanine to serine at position 31 is named H3A31S. A point mutation in the N-terminal tail of H3.3 changing serine to alanine at position 31 is named H3.3S31A. ( C, D ) Overpopulation of the early-stage germ cells in H3A31S-expressing testes compared to H3-expressing testes, both from males aged at 29 degrees for 20 days. The early-stage germ cells region is demonstrated by the brackets, and an overproliferative mitotic spermatogonial cyst is depicted by dotted outline. Immunostaining using anti-Vasa (red), DAPI (blue), anti-α-Spectrin (magenta). Asterisk: niche. Scale bars: 20 μm. ( E ) Quantification of GSCs in H3-expressing testes (n = 30, 8.6 ± 0.3) and H3A31S-expressing testes (n = 30, 14.9 ± 0.8). Unpaired t test to compare the 2 individual datasets to each other. ****: P < 0.0001. Error bars represent the SEM. ( F ) Quantification of early germ cell tumors in H3- and H3A31S-expressing testes. H3 exhibited no tumors (ratio = 0/30) and 30% of H3A31S-expressing testes exhibited early germ cell tumors (ratio = 9/30). ( G ) Expanded hub area in H3A31S-expressing testis compared to H3-expressing testis. The hub areas are depicted by the dotted outline. Individual white dots indicate GSCs. Scale bars: 5 μm. ( H ) Quantification of the hub area of H3- and H3A31S-expressing testes. H3 (n = 30, 93.6 ± 3.8 μm 2 ) and H3A31S (n = 30, 258.1 ± 31.3 μm 2 ) expressing testes. Unpaired t test to compare the 2 individual datasets to each other. ****: P < 0.0001. All data are Avg ± SEM. Error bars represent the SEM. P < 0.0001. The data underlying ( E , F , H ) can be found in S1 Table . CySC, cyst stem cell; GB, gonialblast; GSC, germline stem cell; SEM, standard error of the mean; SG, spermatogonial cell; WT, wild-type.

Drosophila melanogaster gametogenesis represents an ideal model system to study mechanisms regulating the maintenance and proliferation of adult stem cells, as well as proper differentiation of stem and progenitor cells [ 24 , 25 ]. In Drosophila, both male and female germline stem cells (GSCs) can undergo ACD to give rise to self-renewed stem daughter cells and the other daughter cells, which give rise to mature gametes upon differentiation. Male GSCs attach to a group of postmitotic somatic cells called hub cells. A male GSC divides asymmetrically to give rise to both a self-renewed GSC and a gonialblast (GB), the daughter cell that initiates proliferation followed by meiosis and terminal differentiation to become sperm. GBs first go through a transit-amplifying stage with 4 rounds of mitosis as spermatogonial cells (SGs). Once spermatogonial proliferation is complete, cells enter the spermatocyte stage when they initiate a robust gene expression program and epigenomic changes to prepare for meiotic divisions and spermatid differentiation [ 25 – 28 ] ( Fig 1A ).

In eukaryotes, epigenetic mechanisms play a critical role in defining cell identities and functions. There are 2 types of histone proteins: Canonical nucleosomal core histones are mainly incorporated during DNA replication (i.e., H3, H4, H2A, and H2B) [ 12 ]; histone variants are incorporated in a replication-independent manner [ 13 ]. Epigenetic mechanisms such as DNA methylation, posttranslational modifications (PTMs) of histones, histone variants, and chromatin remodeling can instruct cells with identical genomes to turn on different sets of genes and take on distinct identities [ 14 – 18 ]. However, in multicellular organisms, how epigenetic information is maintained or changed during cell divisions, in particular through ACDs, to give rise to daughter cells with distinct cellular fates remained largely unclear [ 19 – 23 ].

Adult stem cells have the unique capability of self-renewal and the ability to differentiate. This balance could be achieved by asymmetric cell division (ACD), which gives rise to 2 daughter cells with distinct fates, one daughter cell with the ability to self-renew and the other daughter cell that is committed to differentiation. ACD occurs widely during development as well as tissue homeostasis and regeneration [ 1 – 5 ]. Imbalance between self-renewal versus differentiation of adult stem cells can result in cancer, tissue degeneration, infertility, as well as aging [ 6 – 11 ].

Results

Rapid turnover of old H3.3S31A compared to WT H3.3 in G2-phase GSCs On the other hand, because the histone variant H3.3 is known to be incorporated in a replication-independent manner [71], we hypothesize that the 31st residue at the N-terminal tail of H3.3 could be important for replication-independent histone turnover during G2 phase. To examine the turnover rate of H3.3 versus H3.3S31A, we measured the change in labeled old histone signals at 12 hours, 24 hours, and 36 hours, corresponding to the first, second, and third cell cycles following the heat shock–induced switch from eGFP- to mCherry-labeled histone expression in GSCs (Fig 6A) [29,30]. In order to pinpoint G 2 phase GSCs, given the very short G 1 phase in GSCs, we used anti-H3S10 phosphorylation (an M-phase marker) and EdU pulse labeling (an S-phase marker) to eliminate nuclei at M-phase or S-phase, respectively [54]. By measuring the eGFP fluorescent signals at each corresponding time points post-heat shock and normalizing them to the no-heat shock control, we found that the levels of old H3.3S31A decreased at a faster rate compared to WT H3.3 (Fig 6B and 6C), with almost all old H3.3S31A histone being turned over by 24 hours (at approximately 2 cell cycles) post-heat shock (Fig 6C and 6D–6D’). These results based on fixed samples are consistent with results using live cell imaging (S5 Fig). Together, these findings suggest the 31st amino acid plays a role in the turnover rate of H3.3, with this mutation accelerating replacement of old histone by new histone. PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 6. Turnover of old H3.3 and H3.3S31A histones in G2 phase GSCs at different time points post-heat shock. (A) Diagram of the time-course recovery experiment (12, 24, and 36 hours) post-heat shock to examine old histones in GSCs. A 30-minute EdU pulse incorporation was performed following the recovery period and right before tissue fixation. G2 phase GSCs were identified by elimination using M-phase (anti-H3S10ph) and S-phase (EdU) markers. (B, C) Confocal images of old histone distribution in (B) H3.3 and (C) H3.3S31A G2 phase GSCs. Asterisk: niche. Scale bars: 20 μm. (D) Quantification of old histone intensity in G2 phase GSCs at each time point post-heat shock recovery. Each data point is representative of average intensity at each time point for H3.3-expressing (12 hours: n = 13, 42.44 ±2.24; 24 hours: n = 13, 26.08 ± 1.77; 36 hours: n = 18, 1.79 ± 0.43) and H3.3S31A-expressing (12 hours: n = 14, 15.06 ± 1.93; 24 hours: n = 15, 0.86 ± 0.25; 36 hours: n = 12, 0.60 ± 0.04) GSCs, measurement from individual GSCs are shown in (D’). Error bars represent the SEM. The data underlying (D, D’) can be found in S6 Table. GSC, germline stem cell; SEM, standard error of the mean. https://doi.org/10.1371/journal.pbio.3002098.g006

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[1] Url: https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3002098

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