Bhalla, Needhi (2021) International Worm Meeting "Strategies to improve equity in faculty hiring"

Bhalla, Needhi

[International Worm Meeting, 2021]

Through targeted recruitment and interventions to support their success during training, the fraction of trainees (graduate students and postdoctoral fellows) in academic science from historically underrepresented groups has steadily increased. However, this trend has not translated to a concomitant increase in the number of faculty from these underrepresented groups. Here, I focus on proven strategies that departments and research institutions can develop to increase equity in faculty hiring and promotion to address the lack of racial and gender diversity among their faculty.

Needhi Bhalla et al. (2005) International Worm Meeting "A Conserved Meiotic Checkpoint Monitors Chromosome Synapsis"

Needhi Bhalla, Abby F Dernburg

[International Worm Meeting, 2005]

Meiosis generates haploid gametes from a diploid cell by coupling a single round of replication with two successive rounds of chromosome segregation. Errors in chromosome segregation during meiosis result in aneuploid gametes and inviable embryos. In humans, certain anueploidies are non-lethal but result in serious developmental disorders, such as Down syndrome. Critical events in meiotic prophase, including the pairing and synapsis of homologous chromosomes and crossover recombination, normally ensure that each gamete contains the correct chromosome complement. We are studying the mechanisms that monitor these events and contribute to their accuracy. Previous work in C. elegans and other organisms has shown that DNA damage that is not repaired during meiotic recombination can target meiotic nuclei for apoptosis. We have now found evidence for a second checkpoint that leads to apoptosis in the germline: errors in synapsis, even in the absence of DNA damage, can act as a direct trigger for cell death. When we analyze mutants where one pair of chromosomes fail to synapse or all chromosomes exhibit asynapsis, we observe elevated levels of germline apoptosis. This activation of apoptosis in response to unsynapsed chromosomes is independent of double-strand break (DSB) formation and DNA damage checkpoint activation, distinguishing it from the pachytene or meiotic recombination checkpoint in budding yeast. However, the C. elegans homolog of the yeast checkpoint component PCH2, is required for synapsis checkpoint activation, suggesting that the molecular mechanism that detects unsynapsed homologous chromosomes is conserved. Our investigations also reveal that at least one of the unsynapsed chromosomes must have a functional Pairing Center (PC) to elicit the checkpoint response. That the PC, a cis-acting site near one end of each chromosome required for synapsis initiation, plays a fundamental role in synapsis checkpoint activation is reminiscent of the role of the kinetochore in spindle assembly checkpoint activation and may reflect an evolutionary relationship between PCs and centromeres. We are interested in identifying additional components of this novel checkpoint to gain a better understanding of how defects in synapsis are monitored and result in apoptosis.

Needhi Bhalla et al. (2003) International Worm Meeting "The Identification of Genes Involved in Meiotic Pairing Center Function"

Needhi Bhalla, Abby F Dernburg

[International Worm Meeting, 2003]

Meiosis is essential for all sexually reproducing organisms. The production of haploid gametes with the correct chromosome complement ensures the restoration of diploidy at fertilization. The pairing and synapsis of homologous chromosomes and subsequent inter-homolog recombination, events that occur in meiotic prophase, are critical requirements for proper meiotic chromosome disjunction. Genetic studies of meiotic recombination in C. elegans have implicated specific chromosome regions as potential pairing centers (PCs): cis-acting sites that ensure appropriate homolog interactions. To gain additional insight into PC function, I am undertaking a RNAi based screen to identify trans-acting factors required for PC function. However, a standard RNAi Him screen will identify general meiotic and chromosome segregation factors in addition to any proteins involved with the PC. Therefore, to enrich for genes that specifically interact with the PC, I am using a mutant background in which one X chromosome lacks a PC as a sensitized genetic background already partially compromised for PC activity; these mutants produce 5-7% male progeny (hermaphrodites in which both X chromosomes lack a PC produce 35%). Animals heterozygous for the deficiency are fed E. coli expressing specific RNAi constructs to see if inactivation of any genes enhance the weak Him phenotype. Reconstruction experiments with RNAi constructs that inactivate him-3 and its homolog, F57C9.5, validate this approach to identifying genes required for pairing and synapsis. Employing cytology and genetics, candidate genes are currently being investigated for roles in PC function.

Devigne, Alice et al. (2019) International Worm Meeting "Do spindle checkpoint components and PCH-2 monitor chromosome movement to regulate and monitor synapsis?"

Bhalla, Needhi, Devigne, Alice

[International Worm Meeting, 2019]

In C. elegans, meiotic nuclei are arrayed in a spatiotemporal gradient in the germline that can be divided in six zones of equal size, allowing us to follow progression of meiotic events, such as pairing, synapsis and recombination, at each point in the germline. During meiotic prophase in C. elegans, chromosomes attach to the nuclear envelope through their Pairing Center (PC), cis acting regions near the end of each chromosome that are essential for pairing and synapsis. This attachment provides access to cytoplasmic elements such as dynein and microtubules that mobilize chromosomes. Recent work in the lab shown that synapsis is regulated by two independent mechanisms: spindle checkpoint proteins and PCH-2. However, it is unclear how these mechanisms are integrated or take advantage of chromosome mobility during their regulation of synapsis. Chromosomes appear to undergo two kinds of displacements during meiotic prophase in order to regulate pairing and synapsis: Processive chromosome motion (PCM), characterized by a continuous motion in the same direction for at least 2 seconds, interspersed with periods in which chromosomes constantly change direction and remaining close to their origin. PCMs are not required for homolog pairing and reduce in frequency when synapsis is complete, suggesting a potential role in regulating synapsis. Here, preliminary data suggests that in mad-1 and bub-3 mutants, PCMs are not abolished but occur at lower frequency. In pch-2 mutants, PCMs are affected differently, exhibiting much less coordinated movement. Actually, we observe PCMs occurring at lower, higher or normal frequency as compared in wild type strain. The difference of PCMs behavior between mad-1 and bub-3 mutants versus pch-2 mutants reinforce the idea that synapsis is regulated by spindle checkpoint components. These three mutants exhibit non-homologous synapsis at very low frequency, but while these events predominate in late pachytene in mad-1 and bub-3 mutants, they occur in early pachytene in pch-2 mutants. When we abolish germline apoptosis, the frequency of non-homologous synapsis increases in mad-1 and pch-2 mutants in late pachytene. However, in bub-3 mutants, we observe more nuclei with asynapsis, both in early and late pachytene, and fewer nuclei with non-homologous synapsis. Thus, while mad-1 and bub-3 are thought to act in the same pathway, they produce slightly different phenotypes with respect to the regulation of phenotype.

Paschal, Cate Randall et al. (2011) International Worm Meeting "ZHP-3 is a ubiquitin E3 ligase with multiple roles in meiosis."

Nelson, Christian, Paschal, Cate Randall, Bhalla, Needhi

[International Worm Meeting, 2011]

During meiotic cell division, homologous chromosomes must pair and undergo crossover recombination, producing a physical linkage that promotes proper chromosome segregation. Recombination is accompanied by a structural reorganization of chromosomes around the crossover site to facilitate segregation on the Meiosis I spindle. In C. elegans, ZHP-3 is required for both crossover formation and for the proper restructuring of chromosomes in late prophase (Jantsch et al., 2004; Bhalla et al., 2008). The presence of a conserved RING finger motif in ZHP-3 suggested that it might act as a ubiquitin E3 ligase and modify substrates to alter their stability, localization, and/or activity. To characterize the ubiquitin ligase activity of ZHP-3, several experiments have been conducted. First, ZHP-3 purified from bacteria shows auto-ubiquitination activity. In addition, a ZHP-3 transgenic lines was generated by microparticle bombardment to facilitate purification of ZHP-3 from adult worms. The construct was designed with zhp-3 genomic sequence under control of the pie-1 promoter, with an N-terminal mCherry-TEV-S (mCherry::zhp-3) tag. The mCherry::zhp-3 transgene can fully rescue the phenotype of the zhp-3(jf61) null allele, recapitulating the localization pattern of the endogenous protein, localizing along the synaptonemal complex in pachytene and as foci in diplotene. The transgenic line displays wildtype levels of viability at 15deg and 20deg C. At 25deg C, there is a slight decrease in viability and an increase in the frequency of male self-progeny, which may be due to elevated expression of the transgene at higher temperatures. The mCherry::zhp-3 strain undergoes crossover recombination, as indicated by the presence of 6 DAPI-staining bivalents at diakinesis. Bivalent chromosomes are properly structured, with HTP-1 localization restricted to the long arm of the bivalent. These data indicate that the mCherry::zhp-3 transgene is competent to carry out both of ZHP-3's roles. In agreement with experiments performed with recombinant protein, mCherry-ZHP-3 immunopurified from adult worm lysate had ubiquitination activity. Mass spectrometry analysis will be performed to identify proteins that co-immunopurify with mCherry-ZHP-3, including targets of its ubiquitination activity. Additionally, candidate substrates will be in vitro transcribed and translated and included in the in vitro ubiquitination reaction. Together, these data help elucidate how ZHP-3's ubiquitin ligase activity facilitates the large-scale changes in chromosome structure in late prophase that are required for proper meiotic chromosome segregation.

Randall, Catherine L. et al. (2009) International Worm Meeting "ZHP-3 is regulated by multiple pathways during meiotic prophase."

Bhalla, Needhi, Randall, Catherine L.

[International Worm Meeting, 2009]

The faithful segregation of chromosomes is tightly regulated to prevent aneuploidy. This is particularly true during meiosis: Defects in meiotic chromosome segregation can produce aneuploid embryos that are typically inviable but can also result in developmental disorders, such as Down syndrome. To ensure proper chromosome segregation during meiosis, homologous chromosomes pair, synapse, and undergo crossover recombination, resulting in their physical linkage. The homolog pair is then dramatically restructured around the crossover to generate a bivalent functional for meiotic chromosome segregation. Recent work has demonstrated that ZHP-3 has two distinct roles during meiotic prophase. First, it is required to promote crossover formation during early pachytene. Additionally, ZHP-3 is required for proper remodeling of the chromosomes to form a functional bivalent. In line with its multiple functions, ZHP-3 undergoes a dramatic relocalization during prophase. It is first localized along the synaptonemal complex (SC) in pachytene. Prior to SC disassembly, ZHP-3 localization becomes restricted to one part of the chromosome. Finally, in late pachytene and diplotene, ZHP-3 is restricted to a single focus on each pair of homologs which marks the site of the crossover. It remains unclear how ZHP-3 is regulated to complete its distinct functions and what controls its relocalization during late prophase. Several independent experiments are being performed to shed light on these mechanisms. First, a non-complementation screen is being performed to isolate zhp-3 alleles with point mutations that specifically disrupt the late prophase role of ZHP-3. We expect that sequencing of these alleles will reveal residues important for the regulation of ZHP-3 in late prophase and provide insight into how ZHP-3 function is modulated to accomplish its two roles. In addition, we have found that ZHP-3 is regulated by the MAP Kinase (MAPK) pathway. The MAPK signaling cascade is required for a switch in the mode of double strand break repair, coincident with the restriction in ZHP-3 localization. Further, the asymmetric distribution of ZHP-3 is lost in mpk-1(ga111ts) strain at the restrictive temperature. We are testing for a genetic interaction between zhp-3 and mpk-1 by evaluating the phenotype of double mutants. Another intriguing feature of ZHP-3 is the presence of a conserved RING finger motif (RFM), a hallmark of ubiquitin E3 ligases. Experiments are underway to determine if ZHP-3 modifies meiotic proteins by the addition of ubiquitin. Together, these experiments will further our understanding of how ZHP-3 is regulated and how it acts to promote accurate chromosome segregation during meiotic cell division.

Lamelza, Piero et al. (2011) International Worm Meeting "Determining Whether Autosomes and the X Chromosome have Distinct Genetic Requirements for Synapsis Checkpoint Activation in C. elegans."

Bhalla, Needhi, Lamelza, Piero

[International Worm Meeting, 2011]

Meiosis is a specialized cell division in which diploid cells give rise to haploid gametes by undertaking a single round of replication and two rounds of chromosome segregation. During meiosis, chromosomes pair, synapse and recombine to generate the chiasmata necessary for proper chromosome segregation. Pairing Centers (PCs), cis-acting sites toward the end of each chromosome, promote pairing and synapsis. Checkpoints monitor proper prophase I progression, curbing the production of aneuploid oocytes by inducing apoptosis of nuclei with meiotic defects. We study the synapsis checkpoint, which is activated by unsynapsed chromosomes bearing a PC and results in increased germline apoptosis. Checkpoint activation occurs when the X-chromosome is unsynapsed (meDf2 heterozygotes) or when all chromosomes are unsynapsed (syp-1 mutants). MES-4, a histone methyl-transferase (HMT) which lays down active H3K36me3 marks, localizes on autosomes and the PC of the X-chromosome. Mutation of mes-4 abolishes the checkpoint when the X is unsynapsed. In contrast, syp-1 mutants require the mutation of another H3K36me3 HMT, MET-1, in conjunction with mes-4 for decreased levels of apoptosis. My project aims to determine whether autosomes and the X-chromosome have different genetic requirements for synapsis checkpoint activation. The model predicts MES-4 is needed for checkpoint activation if the X is unsynapsed while MET-1 is needed for checkpoint activation if the autosomes are unsynapsed. We predict that the active methyl marks laid down by MES-4 and MET-1 are required at PCs to maintain active chromatin on unsynapsed chromosomes which would normally be silenced by meiotic silencing of unpaired chromatin (MSUC).

Hwang, Tom et al. (2013) International Worm Meeting "ZTF-15 is required for the meiotic synapsis checkpoint in C. elegans."

Hwang, Tom, Bhalla, Needhi, Ragle, Matt

[International Worm Meeting, 2013]

In order to achieve proper meiotic chromosome segregation, homologous chromosomes must pair and synapse in prophase I to facilitate crossover recombination. Defects in meiotic prophase events can result in programmed cell death (apoptosis) or cell cycle arrest, indicating that these events are monitored by checkpoint mechanisms to avoid the production of aneuploid gametes. In C. elegans, unsynapsed chromosomes activate germline apoptosis independently of a DNA damage checkpoint that monitors recombination. While independent of one another, both of these checkpoints require the pro-apoptotic factor, egl-1. In response to asynapsis or DNA damage, egl-1 is transcriptionally upregulated to promote apoptosis so that defective meiotic nuclei are removed and do not go on to form aneuploid gametes. When the DNA damage checkpoint is activated, the C. elegans ortholog of tumor suppressor p53, cep-1, is responsible for promoting transcription of egl-1. However, cep-1 is not required for the synapsis checkpoint. We are interested in how egl-1 transcription is upregulated when asynapsis occurs. In an RNAi screen to identify new checkpoint components, we discovered ztf-15 is required for the synapsis checkpoint. ZTF-15 contains 5 C2H2 zinc finger domains, a motif that often mediates binding to specific DNA sequences. We speculate that ZTF-15 is the synapsis checkpoint counterpart of cep-1 and that it acts as a transcription factor to promote egl-1 transcription in the event of asynapsis. To test this hypothesis, we plan to use MOSSci to fluorescently tag ZTF-15 as well as create an antibody that recognizes ZTF-15 to determine where it localizes in the C. elegans germline. If its localization is consistent with a role in apoptosis, we can further test ztf-15's relationship with egl 1 through use of qPCR and gel mobility shift assays. Through these experiments, we hope to elucidate how meiotic events are monitored by cell cycle checkpoints to protect against chromosome missegregation and aneuploidy, which can contribute to infertility, birth defects, and cancer predisposition.

Bohr, Tisha et al. (2015) International Worm Meeting "Spindle assembly checkpoint proteins regulate and monitor meiotic synapsis in C. elegans."

Bohr, Tisha, Nelson, Christian, Bhalla, Needhi

[International Worm Meeting, 2015]

Homolog synapsis is required for proper meiotic chromosome segregation but how synapsis is initiated to stabilize pairing between homologous chromosomes is poorly understood. In C. elegans, synapsis and a meiotic checkpoint that monitors synapsis depend on Pairing Centers, cis-acting loci that interact with nuclear envelope proteins, such as SUN-1, to access cytoplasmic microtubules. We report that synapsis is negatively regulated by spindle assembly checkpoint components. Specifically, we demonstrate that mutations in mdf-1, mdf-2 and bub-3 suppress synapsis defects of dynein mutants. In addition, we show that these proteins are required for the synapsis checkpoint. Consistent with a role at Pairing Centers, MDF-1 and MDF-2 interact with SUN-1 and localize to the periphery of meiotic nuclei. Furthermore, the requirement for spindle assembly checkpoint proteins in regulating synapsis relies on all chromosomes having functional Pairing Centers. We propose that spindle assembly checkpoint proteins monitor the stability of pairing, or tension, between homologous chromosomes to regulate synapsis and elicit a checkpoint response. Our studies uncover an unexpected link between mechanisms that ensure genomic integrity during mitosis and meiotic prophase.

Harper, Nicola et al. (2010) C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany "PLK-2 is a key regulator of meiotic chromosome synapsis."

Dernburg, Abby, Jover-Gil, Sara, Harper, Nicola, Bhalla, Needhi

[C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany, 2010]

The production of haploid gametes from diploid progenitor cells relies on the partitioning of homologous chromosomes during meiosis. Homologous chromosome pairing, formation of the synaptonemal complex (SC) between homologs (synapsis), and recombination are essential for accurate homolog segregation. Multiple checkpoint mechanisms help to ensure that individual oocyte nuclei successfully complete tn before attempting to divide. Large-scale chromosome reorganization typically accompanies homolog pairing and synapsis. In C. elegans , this reorganization defines the meiotic transition zone, which is extended by mutations that cause one or more chromosome pairs to fail to synapse. We have previously reported that this extension of the transition zone in him-8 or syp-1 mutants requires plk-2 , one of several worm genes that encode Polo-like kinases. Oocyte nuclei with persistent unsynapsed chromosomes also preferentially undergo germline apoptosis (Bhalla and Dernburg, 2005). We recently found that plk-2 is required for asynapsis-dependent apoptosis, indicating that the extended transition zone and apoptosis likely reflect activation of the same checkpoint. Consistent with a role in monitoring chromosome synapsis, PLK-2 localizes to the SC. In addition to abrogating the normal responses to unsynapsed chromosomes, mutations in plk-2 alone result in meiotic segregation defects. We found that in the absence of PLK-2, synapsis occurs less processively, resulting in transition zone exit with partially unsynapsed regions. In addition, plk-2 mutations alter the orderly disassembly of the SC. Our results indicate that PLK-2 is a key regulator of SC dynamics and checkpoint control during meiosis.