Colaiacovo lab: Research

We are investigating the mechanisms underlying germline maintenance and accurate chromosome inheritance during meiosis. Addressing this is of vital importance in order to understand the sources of errors that result in dramatically deleterious outcomes including infertility, miscarriages, birth defects such as Down syndrome and tumorigenesis in humans. Our studies are therefore providing key insights into the molecular basis for the regulation of germline maintenance and meiotic segregation. Specifically, we are applying combined genetic, molecular, cytological and biochemical approaches to:

  1. Understand the mechanisms promoting faithful meiotic chromosome inheritance at the molecular level and their regulation throughout meiotic progression.
  2. Explore meiotic chromosome dynamics. Particularly, the interplay between changes in chromosome configuration/structure during meiosis, and homologous chromosome pairing, synapsis, DNA double-strand break repair and accurate segregation.
  3. Investigate the roles played by histone demethylases in germline maintenance and DNA double-strand break repair.

Caenorhabditis elegans: a model system for studies of meiosis and germline maintenance.

We are addressing these aims in the nematode C. elegans. This is an extremely amenable model system for studies of germ cell maintenance and meiosis, which shares a high degree of conservation with humans. The germline accounts for more than half of the cells in the adult worm and its nuclei are distributed throughout the gonad in a defined order, correlating with the sequential stages of classical meiosis. High-resolution 3-D imaging of meiotic chromosomes can be carried out in the context of a well preserved nuclear architecture, and pairing between homologs can be monitored by fluorescence in situ hybridization (FISH). Microarray technology applied to the C. elegans genome has led to the identification of meiotic gene candidates with germline-enriched expression. Techniques such as RNA-mediated interference (RNAi) and PCR-based screens for deletion alleles allow for assessment of the function of germline-active genes.

Fig 1

Meiosis. Meiosis is a specialized cell division program that results in the formation of haploid gametes from diploid germ cells. This cell division program is essential for the perpetuation of most sexually reproducing species and is critical for generating genetic diversity. The end result of meiosis (reduction of the chromosome complement by half) is accomplished by following a single round of DNA replication with two consecutive rounds of chromosome segregation (meiosis I and II). The partitioning of chromosomes needs to be tightly regulated to ensure that homologous chromosomes accurately segregate away from each other at meiosis I, and sister chromatids segregate away from each other during meiosis II. While meiosis II is similar to a mitotic division, chromosomes face a series of unique challenges during meiosis I. In order to overcome these challenges, chromosomes undergo a series of exquisitely orchestrated steps during meiotic prophase I: (1) pairing with their homologous partners, (2) formation of a "zipper-like" proteinaceous structure known as the synaptonemal complex (SC) between paired and aligned homologous chromosomes, and (3) completion of meiotic recombination leading to physical attachments (chiasmata) between homologs. Errors in pairing, synapsis or recombination ultimately lead to chromosome non-disjunction with disastrous consequences for the embryo.

We are applying a multipronged strategy to investigate the molecular basis of mechanisms underlying accurate meiotic chromosome segregation, meiotic chromosome dynamics and germline maintenance. Here are some examples of the projects currently underway in the lab:

Chromosome synapsis

We are investigating the roles and the macromolecular assembly of the synaptonemal complex (SC), a structure at center stage during meiosis, whose functions are poorly understood and a matter of much debate despite its ubiquitous presence from yeast to mammals. Recent identification of three SC components in C. elegans (SYP-1, SYP-2, and SYP-3) coupled with well-established reagents and assays allow us to address the following questions in the lab: How do the SYP proteins assemble to form the SC? What other components are required for synapsis? What are the functions of this structure? Is there a system set up to monitor appropriate assembly and disassembly of the SC?

To address these questions we are examining the interactions among the SC components by biochemical procedures and two-hybrid analysis. We have also succeeded in investigating the organization of the SC by immuno-electron microscopy. In addition, we are identifying new components required for synapsis by continuing our RNAi-based screen for SC components coupled to two-hybrid screens and the identification of co-immunoprecipitated proteins. Ultimately, we will determine the role of candidate SC components in forming the SC structure and their effects on crossover recombination and meiotic progression.

Fig 2

The SC visualized by EM analysis (A) and immunostaining (B) of pachytene nuclei in the C. elegans germline. (A) A continuous stretch of SC can be observed as a "zipper-like" structure flanked by electron-dense patches of chromatin. (B) Immunolocalization of SYP-1 (magenta) places this structural component of the SC at the interface between paired and aligned chromosomes (arrow indicates the SYP-1 signal flanked by the DAPI-stained chromosomes).

Regulation of SC assembly/disassembly and late prophase chromosome remodeling.

We have recently identified and characterized CRA-1, a conserved protein that uncouples chromosome synapsis from recombination and is required for SC formation (Smolikov et al., 2008, PLoS Genetics). Our studies have also revealed that the dynamic process of SC disassembly occurs asymmetrically along the bivalents during late meiotic prophase, and correlates with the single crossover that forms towards the terminal thirds of chromosomes in C. elegans (Nabeshima et al., 2005, J Cell Biol). More recently, we discovered LAB-1, a novel protein required for normal kinetics of SC disassembly and the protection of sister chromatid cohesion (Carvalho et al., 2008, Genes & Development). Taken together, these studies have significantly contributed to our understanding of the process of SC assembly/disassembly. Current and future studies in the lab are aimed at identifying additional components operating in these pathways, understanding the mechanism of function of these proteins, and investigating how chromosome remodeling during late prophase sets the stage for accurate chromosome segregation at meiosis I.

Fig 3

Asymmetric disassembly of the SC. LAB-1 and SYP-1 acquire a reciprocal localization on chromosomes during late pachytene.

Fig 4

Short and Long arm identities observed in bivalents at mid-diakinesis: LAB-1 is observed exclusively on the long arms whereas SYP-1 is present only on the short arms of the bivalent.

Fig 5

LAB-1 forms a distinct ring-like structure on the long arm domain (along with REC-8), presumably delineating the interface between sister chromatids.

DNA double-strand break (DSB) repair in the germline

We are investigating the molecular basis for meiotic crossover formation and germline maintenance through our discovery and analysis of HIM-18/SLX-4, a conserved protein required for the maintenance of genomic integrity given its role in promoting the processing of late homologous recombination intermediates (Saito et al., submitted). Future studies are aimed at investigating the roles played by other proteins implicated along with HIM-18 in DSB repair throughout the germline.

The roles of histone demethylases in germline maintenance and DSB repair

Chromatin structure plays critical roles in most chromosome functions in vivo, including DNA replication, chromosome segregation, and transcription. The roles of histones, the main protein components of chromatin, are controlled to a significant degree by post-translational modifications along their N-terminal tails. One such critical type of modification is histone methylation, which has been implicated in biological processes such as the establishment and maintenance of heterochromatin, transcriptional regulation, X inactivation and DNA damage response. A search for histone tridemethylases in the laboratory of our collaborator, Dr. Yang Shi (Dept. of Pathology, HMS), led to the identification of JMJD2A. This protein is also present in C. elegans and we proceeded to investigate its in vivo biological relevance by focusing on the roles played by CeJMJD2 in the germline. We determined that depletion of this candidate by RNAi leads to an increase in H3-K9Me3 and H3-K36Me3 levels on chromosomes in the adult hermaphrodite germline. This was accompanied by a transient increase in RAD-51 foci and a p53-dependent increase in germ cell apoptosis indicating the activation of a DNA damage checkpoint (Whetstine et al., 2006, Cell). These studies were the first demonstration of the existence of histone demethylases able to reverse lysine trimethylation and have led to an ongoing collaboration through which the link between lysine-specific histone demethylases and DNA repair is being further examined.

Currently, we are analyzing the in vivo roles of spr-5, the lysine specific demethylase LSD1 homolog, in germline maintenance in C. elegans. Through combined genetic and cytological approaches we observed alterations in both histone methylation patterns along germline chromosomes and the progression of DSB repair in spr-5 mutants. We are applying combined genetic, molecular, biochemical and cytological approaches to confirm the in vitro enzymatic activities of SPR-5, investigate its roles in telomere maintenance, DNA damage repair and transcriptional regulation.