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Research Directions

Epigenetic gene regulation in spermatogenesis from stem cells to sperm

To understand the epigenetic principles that govern gene expression programs and the identity of the germline, my laboratory seeks to reveal mechanisms that are essential for establishing the global epigenome in germ cells from stem cells to sperm. We have focused on how the epigenome is preprogrammed in the stem cell stage, which ensures stage-specific transcriptional regulation essential for later stages in spermatogenesis. Our recent work demonstrated that, rather than suppressing genes according to its classic, canonical function, Polycomb Repressive Complex 1 (PRC1) activates genes in spermatogonia, a heterogenous group of cells that include a stem cell population (Maezawa, Genes Dev, 2017). We also discovered that a Polycomb protein specific to the germline, SCML2, forms a complex with PRC1 to establish a silent epigenetic memory on somatic genes in undifferentiated spermatogonia (Hasegawa et.al, Dev Cell, 2015), and this memory manifests in subsequent gene silencing after differentiation. Ongoing experiments aim to understand the epigenetic mechanismsthat sustain long-term maintenance of male fertility.

Maezawa S, Hasegawa K, Yukawa M, Sakashita A, Alavattam KG, Andreassen PR, Vidal M, Koseki H, Barski A, Namekawa SH. Polycomb directs timely activation of germline genes in spermatogenesis. Genes Dev. 2017;31(16):1693-703.

Hasegawa K, Sin HS, Maezawa S, Broering TJ, Kartashov AV, Alavattam KG, Ichijima Y, Zhang F, Bacon WC, Greis KD, Andreassen PR, Barski A, Namekawa SH. SCML2 Establishes the Male Germline Epigenome through Regulation of Histone H2A Ubiquitination. Dev Cell. 2015;32(5):574-88.

The mechanisms in unipotent germ cells that prepare for a totipotent zygote after fertilization

A central question in developmental biology is how differentiated, unipotent germ cells give rise to totipotent zygotes following fertilization. To approach this question, our laboratory aims to elucidate the epigenetic mechanisms that regulate heritable epigenetic information. Our recent work reveals that, in meiotic prophase I, another function of SCML2 is to interact with a separate Polycomb complex, PRC2, to establish bivalent genomic domains, which contain PRC2-mediated repressive H3K27me3 together with active H3K4me2/3 marks (Maezawa et.al, PNAS, 2018). Initially discovered in embryonic stem (ES) cells as a signature of developmental potential, bivalent genomic domains are maintained at several hundred developmental regulator genes in germ cells. These genes remain silent in germline development and, in sperm, histones at bivalent domains are retained as opposed to being replaced with protamines. We have discovered a novel class of bivalent genomic domains on several thousand somatic genes after the mitosis-to-meiosis transition (Sin et.al, BMC Biol, 2015), thus revealing extensive epigenetic programming in meiosis. These results raise the possibility that SCML2-mediated epigenetic memories impact the next generation.

Sin HS, Kartashov AV, Hasegawa K, Barski A, Namekawa SH. Poised chromatin and bivalent domains facilitate the mitosis-to-meiosis transition in the male germline. BMC Biol. 2015;13:53.

Maezawa S, Hasegawa K, Yukawa M, Kubo N, Sakashita A, Alavattam KG, Sin HS, Kartashov AV, Sasaki H, Barski A, Namekawa SH. Polycomb protein SCML2 facilitates H3K27me3 to establish bivalent domains in the male germline. PNAS. 2018;115(19):4957-62.

Reprogramming of high order chromatin and active enhancers in spermatogenesis

The testis has the most diverse, complex, and rapidly evolved transcriptome of any organ. During spermatogenesis, genome-wide gene expression undergoes dynamic, large-scale changes. After the mitosis-to-meiosis transition, several thousand spermatogenesis-specific genes are activated while, at the same time, thousands of genes expressed in both somatic lineages and progenitor cells of the germline are suppressed. During spermatogenesis, progressive, dynamic chromatin remodeling takes place to produce haploid spermatids (Maezawa et.al, J Cell Sci, 2018). Together with genome-wide changes in gene expression, the mitosis-to-meiosis transition accompanies dynamic reorganization of epigenome, accessible chromatin, active enhancers, retrotransposon activity, and 3D chromatin conformation (Sin et.al, BMC Biol, 2015, Maezawa et.al, Nucleic Acids Res, 2018, Alavattam et.al, Nat Struct Mol Biol, 2019, Maezawa et.al, Nat Struct Mol Biol, 2020, Sakashita et.al, Nat Struct Mol Biol, 2020), —all in preparation for the next generation of life.

We recently revealed the 3D organization of chromatin in male germ cells during spermatogenesis using Hi-C analysis (Alavattam et.al, Nat Struct Mol Biol, 2019). Our data indicate that male meiosis occurs amid global reprogramming of 3D chromatin organization, and we found that chromatin compartments become stronger in spermiogenesis, a putative requirement in preparing the next generation of life. As well, we determined the profiles of active enhancers in representative stages of mouse spermatogenesis, and we identified a meiotic type of super-enhancer (SE), demonstrating that the dynamic transcriptome change at the mitosis-to-meiosis transition is driven by a switch from mitotic to meiotic types of SEs (Maezawa et.al, Nat Struct Mol Biol, 2020).

In mammals, germline transcriptomes are highly divergent—a crucial feature of the germline and a hallmark feature of speciation. However, the mechanisms that define species-specific germline transcriptomes remain undetermined. In our recent work, we show that evolutionarily young endogenous retroviruses (ERVs) function as species-specific enhancers (Sakashita et.al, Nat Struct Mol Biol, 2020). These enhancers drive evolutionarily novel germline genes after the mitosis-to-meiosis transition, thereby defining species-specific germline transcriptomes among mammals. In humans, independently evolved ERVs are associated with the expression of human-specific germline genes, demonstrating the prevalence of ERV-driven mechanisms in mammals (Sakashita et.al, Nat Struct Mol Biol, 2020).

Maezawa S, Hasegawa K, Alavattam KG, Funakoshi M, Sato T, Barski A, Namekawa SH. SCML2 promotes heterochromatin organization in late spermatogenesis. J Cell Sci. 2018;131(17).

Maezawa S, Yukawa M, Alavattam KG, Barski A, Namekawa SH. Dynamic reorganization of open chromatin underlies diverse transcriptomes during spermatogenesis. Nucleic Acids Res. 2018;46(2):593-608.

Alavattam KG, Maezawa S, Sakashita A, Khoury H, Barski A, Kaplan N, Namekawa SH. Attenuated chromatin compartmentalization in meiosis and its maturation in sperm development. Nat Struct Mol Biol. 2019;26:175-184.

Maezawa S, Sakashita A, Yukawa M, Chen X, Takahashi K, Alavattam KG, Nakata I, Weirauch MT, Barski A, Namekawa SH. Super-enhancer switching drives a burst in gene expression at the mitosis-to-meiosis transition. Nat Struct Mol Biol. 2020;27:978-988.

Sakashita A, Maezawa S, Takahashi K, Alavattam KG, Yukawa M, Hu Y-C, Kojima S, Parrish NF, Barski A, Pavlicev M, Namekawa SH. Endogenous retroviruses drive species-specific germline transcriptomes in mammals. Nat Struct Mol Biol. 2020;27:967-977.

DNA damage response pathways in the regulation of the sex chromosomes

Meiosis is a hallmark event in germline development when, before producing haploid gametes, paternal and maternal chromosomes undergo synapsis and the genomic shuffling that is recombination. The fidelity of these processes is strictly monitored by a meiotic checkpoint. However, the molecular basis of this checkpoint, which serves to safeguard the germline, remains unknown.

In mammalian male meiosis, the X and Y chromosomes are subject to meiotic sex chromosome inactivation (MSCI). MSCI is an essential process in the male germline: Failure to initiate MSCI is linked to the complete arrest and elimination of male germ cells in the pachytene stage of meiotic prophase I. MSCI is a sex chromosome-specific manifestation of meiotic silencing of unsynapsed chromatin, a general mechanism for transcriptional silencing in meiosis that operates as a surveillance mechanism for chromosome asynapsis. We identified essential meiotic functions for DDR pathways: These pathways safeguard the male germline by monitoring unsynapsed chromosomes (Ichijima et.al, Genes Dev, 2011, Sin et.al, Genes Dev, 2012, Broering et.al, J Cell Biol, 2014). We demonstrated that Fanconi Anemia proteins are critical epigenetic regulators in MSCI (Kato et.al, Hum Mol Genet, 2015, Alavattam et.al, Cell Rep, 2016). We also determined the function of CHEK1 in male meiosis (Abe et.al, Hum Mol Genet, 2018).

In another recent study, we provided a clue to solving a long-standing mystery behind MSCI: Our study suggests that the initiation of MSCI sequesters DDR signaling from autosomes to the sex chromosomes; as a result, the establishment of MSCI permits the timely progression of male germ cells through meiotic prophase I (Abe et.al, Curr Biol, 2020).

Kato Y, Alavattam KG, Sin HS, Meetei AR, Pang Q, Andreassen PR, Namekawa SH. FANCB is essential in the male germline and regulates H3K9 methylation on the sex chromosomes during meiosis. Hum Mol Genet. 2015;24(18):5234-49.

Alavattam KG, Kato Y, Sin HS, Maezawa S, Kowalski IJ, Zhang F, Pang Q, Andreassen PR, Namekawa SH. Elucidation of the Fanconi Anemia Protein Network in Meiosis and Its Function in the Regulation of Histone Modifications. Cell Rep. 2016;17(4):1141-57.

Abe H, Alavattam KG, Kato Y, Castrillon DH, Pang Q, Andreassen PR, Namekawa SH. CHEK1 coordinates DNA damage signaling and meiotic progression in the male germline of mice. Hum Mol Genet. 2018;27(7):1136-49.

Abe H, Alavattam KG, Hu YC, Pang Q, Andreassen PR, Hegde RS, Namekawa SH. The Initiation of Meiotic Sex Chromosome Inactivation Sequesters DNA Damage Signaling from Autosomes in Mouse Spermatogenesis. Curr Biol. 2020;30(3):408-20.e5.

Epigenetic gene regulation of the sex chromosomes

Our subsequent and current studies are revealing that the DDR pathway is not only responsible for monitoring asynapsis in meiosis, but also for directing epigenetic gene regulation, both gene silencing and gene activation of the sex chromosomes, in the later stages of germ cells. A breakthrough in our research was the identification of the germline-specific protein SCML2 as a binding protein of silent sex chromosome in meiosis (Hasegawa et.al, Dev Cell, 2015). Our studies are establishing a novel concept of epigenetic programming of the sex chromosomes in the germline and further uncovering the impact of MSCI on the genomic evolution of the sex chromosomes. Following the initiation of MSCI, the X chromosome uniquely establishes open chromatin (Maezawa et.al, Nucleic Acids Res, 2018), active enhancers (Adams et.al, Plos Genet, 2018), and distinct 3D chromatin (Alavattam et.al, Nat Struct Mol Biol, 2019).

Hasegawa K, Sin HS, Maezawa S, Broering TJ, Kartashov AV, Alavattam KG, Ichijima Y, Zhang F, Bacon WC, Greis KD, Andreassen PR, Barski A, Namekawa SH. SCML2 Establishes the Male Germline Epigenome through Regulation of Histone H2A Ubiquitination. Dev Cell. 2015;32(5):574-88.

Adams SR, Maezawa S, Alavattam KG, Abe H, Sakashita A, Shroder M, Broering TJ, Sroga Rios J, Thomas MA, Lin X, Price CM, Barski A, Andreassen PR, Namekawa SH. RNF8 and SCML2 cooperate to regulate ubiquitination and H3K27 acetylation for escape gene activation on the sex chromosomes. PLoS Genet. 2018;14(2):e1007233.

Maezawa S, Yukawa M, Alavattam KG, Barski A, Namekawa SH. Dynamic reorganization of open chromatin underlies diverse transcriptomes during spermatogenesis. Nucleic Acids Res. 2018;46(2):593-608.

Alavattam KG, Maezawa S, Sakashita A, Khoury H, Barski A, Kaplan N, Namekawa SH. Attenuated chromatin compartmentalization in meiosis and its maturation in sperm development. Nat Struct Mol Biol. 2019;26(3):175-84.

Cancer/germline genes and tumor suppressors in the germline

We have identified a critical function for the cancer/germline gene Scml2 in the regulation of the germ cell epigenome (Hasegawa et al. Dev Cell 2015). Typically suppressed in somatic cells, Scml2 is a germline-specific gene that is highly expressed in many cancers. As such, we hypothesize that SCML2 is a critical regulator of a germline program activated in many types of cancers. In addition to our studies of cancer/germline genes, we have determined the fundamental functions of various tumor suppressors in the context of DNA damage response pathways. Those include MDC1 (Ichijima et al. Genes Dev 2011), RNF8 (Sin et al. Genes Dev 2012), BRCA1 (Broering et al. J Cell Biol 2014), FANCB (Kato et al. Hum Mol Genet 2015), several factors in the Fanconi anemia pathway (Alavattam et al. Cell Rep 2016), CHEK1 (Abe et al. Hum Mol Genet 2018), UHRF1 (Dong et al. Nat Commun 2019), BRUCE (Che et al. Cell Death Differ, 2020), and the histone variant H2AX (Abe et al. Curr Biol, 2020).

Hasegawa K, Sin HS, Maezawa S, Broering TJ, Kartashov AV, Alavattam KG, Ichijima Y, Zhang F, Bacon WC, Greis KD, Andreassen PR, Barski A, Namekawa SH. SCML2 Establishes the Male Germline Epigenome through Regulation of Histone H2A Ubiquitination. Dev Cell. 2015;32(5):574-88.

Alavattam KG, Kato Y, Sin HS, Maezawa S, Kowalski IJ, Zhang F, Pang Q, Andreassen PR, Namekawa SH. Elucidation of the Fanconi Anemia Protein Network in Meiosis and Its Function in the Regulation of Histone Modifications. Cell Rep. 2016;17(4):1141-57.

Kato Y, Alavattam KG, Sin HS, Meetei AR, Pang Q, Andreassen PR, Namekawa SH. FANCB is essential in the male germline and regulates H3K9 methylation on the sex chromosomes during meiosis. Hum Mol Genet. 2015;24(18):5234-49.

Abe H, Alavattam KG, Hu YC, Pang Q, Andreassen PR, Hegde RS, Namekawa SH. The Initiation of Meiotic Sex Chromosome Inactivation Sequesters DNA Damage Signaling from Autosomes in Mouse Spermatogenesis. Curr Biol. 2020;30(3):408-20.e5.