Kenneth Lee

• Ph.D.
• Manager at Clinical Reference Laboratory

Eco1 is the acetyltransferase that establishes sister-chromatid cohesion during DNA replication. A budding yeast strain with an eco1 mutation that genocopies Roberts syndrome has reduced ribosomal DNA (rDNA) transcription and a transcriptional signature of starvation. We show that deleting FOB1—a gene that encodes a replication fork-blocking protei...

The cohesin complex contributes to ribosome function, although the molecular mechanisms involved are unclear. Compromised cohesin function is associated with a class of diseases known as cohesinopathies. One cohesinopathy, Roberts syndrome (RBS), occurs when a mutation reduces acetylation of the cohesin Smc3 subunit. Mutation of the cohesin acetylt...

Roberts syndrome (RBS) is a human disease characterized by defects in limb and craniofacial development and growth and mental retardation. RBS is caused by mutations in ESCO2, a gene which encodes an acetyltransferase for the cohesin complex. While the essential role of the cohesin complex in chromosome segregation has been well characterized, it p...

SNO1 and SNZ1 misregulation in cohesin mutants is confirmed by quantitative PCR. RT-qPCR was performed on the RNAs from each timepoint for WT and the two mutants. Gene specific primers for SNZ1 (A) and SNO1 (B) were used and the increase over time was calculated relative to ACT1 and PGK1. Both SNZ1 and SNO1 are more strongly induced in the mutants....

Growth and rRNA labeling. A. RNA was pulse labeled and the incorporation of 3H-uridine was quantified in W303a and eco1-W216G strains as performed in Figure 5. B. The growth of the strains used for (A) in SD-ura at 30°C is shown. C. The growth of the strains indicated is shown in YPD+CSM at 37°C. The growth curve and statistics for B and C were per...

An expanded presentation of the FISH data. A. The standard curve shown was used to determine how fluorescence intensity relates to number of RNAs. The fluorescence for each cell is measured and then binned to show the fraction of the population with each RNA number. The fluorescence for 300 cells from 3 independent cultures for each strain was meas...

qPCR assay for a sequence on the left and right arm of each chromosome confirms that no aneuploidy is present in either the W303a strain or the smc1-Q843Δ (SG136) and eco1-W216G (SG156) mutants derived from this strain. (PDF)

Strains. (XLSX)

Genes adjacent to tDNAs are not misregulated in cohesin mutants. We examined the expression of genes adjacent to tRNAs in the microarray data set. The coordinates for all yeast tRNA genes were retrieved from Ensembl (299 genes). A script was written to use the Ensembl API and select the nearest gene to the left and to the right of each tRNA. The re...

GO analysis of misregulated genes. (XLSX)

Cohesin is a protein complex known for its essential role in chromosome segregation. However, cohesin and associated factors have additional functions in transcription, DNA damage repair, and chromosome condensation. The human cohesinopathy diseases are thought to stem not from defects in chromosome segregation but from gene expression. The role of...

The Saccharomyces cerevisiae SUN-domain protein Mps3 is required for duplication of the yeast centrosome-equivalent organelle, the spindle pole body (SPB), and it is involved in multiple aspects of nuclear organization, including telomere tethering and gene silencing at the nuclear membrane, establishment of sister chromatid cohesion, and repair of...

All dNSAF for all proteins detected in S. cerevisiae Gcn5 HAT complexes Wild-Type following Contaminant Extraction. This table contains the locus identification, description, acronym, open reading frame ID (ORF ID) and dNSAF values for all proteins detected in wild type complex purifications prior to contaminant extraction.

All dNSAF values for all proteins detected in S. cerevisiae Gcn5 HAT complexes in the deletion dataset following contaminant extraction. This table contains the locus identification, description, acronym, open reading frame ID (ORF ID) and dNSAF values for all proteins detected in protein complexes analyzed from deletion strains prior to contaminan...

Supplementary methods, results, Figures 1–6, and Table 10.

All peptides per protein that were identified in our wild-type analysis. This table contains the protein names, spectra names, scoring information, sequence information, and summary information for all peptides identified in the wild type protein complexes analyses.

List of Proteins Detected in S. cerevisiae Gcn5 HAT complexes Wild-Type Prior to Contaminant Extraction. This table contains the locus identification, description, peptide counts, spectral count, shared spectral count, distributed peptides counts, unique peptide counts, sequence coverage, and distributed normalized spectral abundance factor values...

Interaction data obtained from the SAGA catalytic mutants. This table contains the T locus identification, description, peptide counts, spectral count, shared spectral count, distributed peptides counts, unique peptide counts, sequence coverage, and distributed normalized spectral abundance factor values for all proteins detected in catalytic mutan...

All peptides per protein that were identified in the deletion analysis. This table contains the protein names, spectra names, scoring information, sequence information, and summary information for all peptides identified in the protein complexes analyses from deletion strains.

List of Proteins Detected in S. cerevisiae Gcn5 HAT complexes in the Deletion Dataset Prior to Contaminant Extraction. This table contains the locus identification, description, peptide counts, distributed spectral count, sequence coverage, and distributed normalized spectral abundance factor values for all proteins detected in protein complexes an...

The location of the different subunits of the SAGA/ADA complexes within the distinct modules. This table contains the module defined in previous studies, the subunits assigned in previous studies, the literature support of this, the module definition in our study, and the subunits assigned in our study in order to compare previous results with our...

List of probabilities between each bait and prey in all complexes. This table contains in (A) the list of probabilities between each bait and prey in wild-type Gcn5 HAT complexes and (B) the list of probabilities between each bait and prey in a deletion Gcn5 HAT complexes.

Despite the availability of several large-scale proteomics studies aiming to identify protein interactions on a global scale, little is known about how proteins interact and are organized within macromolecular complexes. Here, we describe a technique that consists of a combination of biochemistry approaches, quantitative proteomics and computationa...

The SAGA (Spt-Ada-Gcn5 acetyltransferase) complex is an important chromatin modifying complex that can both acetylate and deubiquitinate histones. Sgf29 is a novel component of the SAGA complex. Here, we report the crystal structures of the tandem Tudor domains of Saccharomyces cerevisiae and human Sgf29 and their complexes with H3K4me2 and H3K4me3...

Trimethyl-lysine (me3) modifications on histones are the most stable epigenetic marks and they control chromatin-mediated regulation of gene expression. Here, we determine proteins that bind these marks by high-accuracy, quantitative mass spectrometry. These chromatin "readers" are assigned to complexes by interaction proteomics of full-length BAC-...

Spinocerebellar ataxia (SCA) is a physically devastating, genetically inherited disorder characterized by abnormal brain function that results in the progressive loss of the ability to coordinate movements. There are many types of SCAs as there are various gene mutations that can cause this disease. SCA types 1–3, 6–10, 12, and 17 result from a tri...

Figure S2 Description: Sgf73 provided in trans is able to incorporate into the SAGA/SLiK (SALSA) complex and partially rescue histone deubiquitination activity. A. Silver stain gel showing purification of SAGA/SLiK(SALSA) from yeast expressing SGF73 from a vector under control of it's own promoter (Lane 1). B. MudPit analysis of the rescued complex...

Figure S1 A. Silver stain of various SAGA/SLiK(SALSA) purifications. Lane 1 marker, Lane 2 Spt8Tap purification, Lane 3 Gcn5TAP;sgf73Δ purification, Lane 4 Gcn5Tap purification, Lane 5 Spt8TAP;sgf73Δ purification, Lane 6 SLiK(SALSA) purification from an Spt7TAP strain, where Spt7 lacks the C-terminus required for Spt8 association, Lane 7 Sgf73TAP p...

Nonstop, which has previously been shown to have homology to ubiquitin proteases, is required for proper termination of axons R1-R6 in the optic lobe of the developing Drosophila eye. Herein, we establish that Nonstop actually functions as an ubiquitin protease to control the levels of ubiquitinated histone H2B in flies. We further establish that N...

Over the past 10 years, the study of histone acetyltransferases (HATs) has advanced significantly, and a number of HATs have been isolated from various organisms. It emerged that HATs are highly diverse and generally contain multiple subunits. The functions of the catalytic subunit depend largely on the context of the other subunits in the complex....

Barrier-to-autointegration factor (BAF) is an essential chromatin protein conserved in metazoans. BAF has roles in nuclear assembly, chromatin organization, gene expression, and gonad development and is exploited by retroviruses. BAF forms stable dimers that bind nonspecifically to dsDNA and specifically to LEM-domain proteins (e.g., LAP2beta, emer...

Barrier to autointegration factor (BAF) is an essential conserved double-stranded DNA-binding protein in metazoans. BAF binds directly to LEM domain nuclear proteins (e.g. LAP2, Emerin, and MAN1), lamin A, homeodomain transcription factors, and human immunodeficiency virus type 1-encoded proteins. BAF influences higher order chromatin structure and...

Yeast Rpd3 histone deacetylase plays an important role at actively transcribed genes. We characterized two distinct Rpd3 complexes, Rpd3L and Rpd3S, by MudPIT analysis. Both complexes shared a three subunit core and Rpd3L contains unique subunits consistent with being a promoter targeted corepressor. Rco1 and Eaf3 were subunits specific to Rpd3S. M...

Covalent modifications of the histone tails and the cross talk between these modifications are hallmark features of gene regulation. The SAGA histone acetyltransferase complex is one of the most well-characterized complexes involved in these covalent modifications. The recent finding that the removal of the ubiquitin group from H2B is performed by...

Epigenetics is the alteration of phenotype without affecting the genotype. An underlying molecular mechanism of epigenetics is the changes of chromatin structure by covalent histone modifications and nucleosome reorganization. In the yeast, Saccharomyces cerevisiae, two of the most well-studied macromolecular complexes that perform these epigenetic...

Caenorhabditis elegans mtf-1 encodes matefin, which has a predicted SUN domain, a coiled-coil region, an anti-erbB-2 IgG domain, and two hydrophobic regions. We show that matefin is a nuclear membrane protein that colocalizes in vivo with Ce-lamin, the single nuclear lamin protein in C. elegans, and binds Ce-lamin in vitro but does not require Ce-l...

LEM-domain proteins share a folded structure, the 'LEM-domain', which binds a conserved chromatin protein named BAF. Most LEM-domain proteins are found at the nuclear membrane, but some are nucleoplasmic. All characterized members of this family bind nuclear lamin filaments. We summarize the 'founding' LEM-domain proteins LAP2, emerin and MAN1 ('SA...

Barrier-to-autointegration factor (BAF) is a conserved human chromatin protein exploited by retroviruses. Previous investigators showed that BAF binds double-stranded DNA nonspecifically and is a host component of preintegration complexes (PICs) isolated from cells infected with human immunodeficiency virus type 1 (HIV-1) or Moloney murine leukemia...

Emerin and MAN1 are LEM domain-containing integral membrane proteins of the vertebrate nuclear envelope. The function of MAN1 is unknown, whereas emerin is known to interact with nuclear lamins, barrier-to-autointegration factor (BAF), nesprin-1 alpha, and a transcription repressor. Mutations in emerin cause X-linked recessive Emery-Dreifuss muscul...

Emerin belongs to the "LEM domain" family of nuclear proteins, which contain a characteristic approximately 40-residue LEM motif. The LEM domain mediates direct binding to barrier to autointegration factor (BAF), a conserved 10-kDa chromatin protein essential for embryogenesis in Caenorhabditis elegans. In mammalian cells, BAF recruits emerin to ch...

Nesprin-1alpha is a spectrin repeat (SR)-containing, transmembrane protein of the inner nuclear membrane, and is highly expressed in muscle cells. A yeast two-hybrid screen for nesprin-1alpha-interacting proteins showed that nesprin-1alpha interacted with itself. Blot overlay experiments revealed that nesprin-1alpha's third SR binds the fifth SR. T...

Mutations in the Caenorhabditis elegans unc-84 gene cause defects in nuclear migration and anchoring. We show that endogenous UNC-84 protein colocalizes with Ce-lamin at the nuclear envelope and that the envelope localization of UNC-84 requires Ce-lamin. We also show that during mitosis, UNC-84 remains at the nuclear periphery until late anaphase,...

Emerin belongs to the LEM-domain family of nuclear membrane proteins, which are conserved in metazoans from C. elegans to humans. Loss of emerin in humans causes the X-linked form of Emery-Dreifuss muscular dystrophy (EDMD), but the disease mechanism is not understood. We have begun to address the function of emerin in C. elegans, a genetically tra...

Loss of emerin, a lamin-binding nuclear membrane protein, causes Emery-Dreifuss muscular dystrophy. We analyzed 13 site-directed mutations, and four disease-causing mutations that do not disrupt emerin stability or localization. We show that emerin binds directly to barrier-to-autointegration factor (BAF), a DNA-bridging protein, and that this bind...

Mutations in emerin cause the X-linked recessive form of Emery-Dreifuss muscular dystrophy (EDMD). Emerin localizes at the inner membrane of the nuclear envelope (NE) during interphase, and diffuses into the ER when the NE disassembles during mitosis. We analyzed the recruitment of wildtype and mutant GFP-tagged emerin proteins during nuclear envel...

LAP2 belongs to a family of nuclear membrane proteins sharing a 43 residue LEM domain. All LAP2 isoforms have the same N-terminal ‘constant’ region (LAP2-c), which includes the LEM domain, plus a C-terminal ‘variable’ region. LAP2-c polypeptide inhibits nuclear assembly in Xenopus extracts, and binds in vitro to barrier-to-autointegration factor (B...

The nuclear lamina mediates nuclear structure, chromatin organization, and chromosome segregation, and may have structural roles in transcription and the elongation phase of DNA replication. The lamina also retains nuclear membrane proteins, determines nuclear shape, and controls the spatial distribution of nuclear pore complexes. This astonishing...

The number and complexity of genes encoding nuclear lamina proteins has increased during metazoan evolution. Emerging evidence reveals that transcriptional repressors such as the retinoblastoma protein, and apoptotic regulators such as CED-4, have functional and dynamic interactions with the lamina. The discovery that mutations in nuclear lamina pr...

Emerin, MAN1, and LAP2 are integral membrane proteins of the vertebrate nuclear envelope. They share a 43-residue N-terminal motif termed the LEM domain. We found three putative LEM domain genes in Caenorhabditis elegans, designated emr-1, lem-2, and lem-3. We analyzed emr-l, which encodes Ce-emerin, and lem-2, which encodes Ce-MAN1. Ce-emerin and...

The HIR1 gene product is required to repress transcription of three of the four histone gene loci in Saccharomyces cerivisiae, and like its counterpart, the HIR2 protein, it functions as a transcriptional corepressor. Although Hir1p and Hir2p are physically associated in yeast, Hir1p is able to function independently of Hir2p when it is artificiall...

The HIR/HPC (histone regulation/histone periodic control) negative regulators play important roles in the transcription of six of the eight core histone genes during the Saccharomyces cerevisiae cell cycle. The phenotypes of hir1 and hir2 mutants suggested that the wild-type HIR1 and HIR2 genes encode transcriptional repressors that function in the...