Dallan Young

Three Yeast Proteins Related to the Human Candidate Tumor Suppressor p33ING1 Are Associated with Histone Acetyltransferase Activities

ABSTRACT Three Saccharomyces cerevisiae proteins (Yng1/YOR064c, Yng2/YHR090c, and Pho23) and two Schizosaccharomyces pombeproteins (Png1/CAA15917 and Png2/CAA21250) share significant sequence identity with the human candidate tumor suppressor p33ING1in their C-terminal regions. The homologous regions contain PHD finger domains which have been implicated in chromatin-mediated transcriptional regulation. We show that GFP-Yng2, like human Ing1, is localized in the nucleus. Deletion of YNG2 results in several phenotypes, including an abnormal multibudded morphology, an inability to utilize nonfermentable carbon sources, heat shock sensitivity, slow growth, temperature sensitivity, and sensitivity to caffeine. These phenotypes are suppressed by expression of either human Ing1 or S. pombe Png1, suggesting that the yeast and human proteins are functionally conserved. Yng1- and Pho23-deficient cells also share some of these phenotypes. We demonstrated by yeast two-hybrid and coimmunoprecipitation tests that Yng2 interacts with Tra1, a component of histone acetyltransferase (HAT) complexes. We further demonstrated by coimmunoprecipitation that HA-Yng1, HA-Yng2, HA-Pho23, and HA-Ing1 are associated with HAT activities in yeast. Genetic and biochemical evidence indicate that the Yng2-associated HAT is Esa1, suggesting that Yng2 is a component of the NuA4 HAT complex. These studies suggest that the yeast Ing1-related proteins are involved in chromatin remodeling. They further suggest that these functions may be conserved in mammals and provide a possible mechanism for the human Ing1 candidate tumor suppressor.

Human ING1 Proteins Differentially Regulate Histone Acetylation

ING1 proteins are nuclear, growth inhibitory, and regulate apoptosis in different experimental systems. Here we show that similar to their yeast homologs, human ING1 proteins interact with proteins associated with histone acetyltransferase (HAT) activity, such as TRRAP, PCAF, CBP, and p300. Human ING1 immunocomplexes contain HAT activity, and overexpression of p33ING1b, but not of p47ING1a, induces hyperacetylation of histones H3 and H4, in vitro and in vivo at the single cell level. p47ING1a inhibits histone acetylation in vitro and in vivo and binds the histone deacetylase HDAC1. Finally, we present evidence indicating that p33ING1b affects the degree of physical association between proliferating cell nuclear antigen (PCNA) and p300, an association that has been proposed to link DNA repair to chromatin remodeling. Together with the finding that human ING1 proteins bind PCNA in a DNA damage-dependent manner, these data suggest that ING1 proteins provide a direct linkage between DNA repair, apoptosis, and chromatin remodeling via multiple HAT·ING1·PCNA protein complexes.

Identification of a human cDNA encoding a protein that is structurally and functionally related to the yeast adenylyl cyclase-associated CAP proteins

The adenylyl cyclases of both Saccharomyces cerevisiae and Schizosaccharomyces pombe are associated with related proteins named CAP. In S. cerevisiae, CAP is required for cellular responses mediated by the RAS/cyclic AMP pathway. Both yeast CAPs appear to be bifunctional proteins: the N-terminal domains are required for the proper function of adenylyl cyclase, while loss of the C-terminal domains results in morphological and nutritional defects that appear to be unrelated to the cAMP pathways. Expression of either yeast CAP in the heterologous yeast suppresses phenotypes associated with loss of the C-terminal domain of the endogenous CAP but does not suppress loss of the N-terminal domain. On the basis of the homology between the two yeast CAP proteins, we have designed degenerate oligonucleotides that we used to detect, by the polymerase chain reaction method, a human cDNA fragment encoding a CAP-related peptide. Using the polymerase chain reaction fragment as a probe, we isolated a human cDNA clone encoding a 475-amino-acid protein that is homologous to the yeast CAP proteins. Expression of the human CAP protein in S. cerevisiae suppresses the phenotypes associated with loss of the C-terminal domain of CAP but does not suppress phenotypes associated with loss of the N-terminal domain. Thus, CAP proteins have been structurally and, to some extent, functionally conserved in evolution between yeasts and mammals.

Mkh1, a MEK Kinase Required for Cell Wall Integrity and Proper Response to Osmotic and Temperature Stress in Schizosaccharomyces pombe

We have identified a Schizosaccharomyces pombe gene, mkh1, that encodes a MEK kinase (MEKK) homolog. The coding region of mkh1 is contained within a single exon encoding a 1,116-amino-acid protein. The putative catalytic domain of Mkh1 is 54% identical to the catalytic domain of S. cerevisiae Bck1, the most closely related protein. Deletion of mkh1 did not significantly affect cell growth or division under standard conditions. However, mkh1delta cell growth was inhibited by high KCl or NaCl concentrations. mkh1delta cells required a longer time to reenter the cell cycle after prolonged stationary-phase arrest. Also, mkh1delta cells exhibited a round cell shape, while overexpression of Mkh1 resulted in an elongated cell shape. mkh1delta cells exhibited a more dramatic phenotype when grown in nutrient-limiting conditions at high temperature or in hyperosmotic medium. In such conditions, completion of cytokinesis was inhibited, resulting in the growth of pseudohyphal filaments with multiple septa and nuclei. Also, mkh1delta cells were hypersensitive to beta-glucanase treatment. Together these results suggest that Mkh1 regulates cell morphology, cell wall integrity, salt resistance, cell cycle reentry from stationary-phase arrest, and filamentous growth in response to stress. These phenotypes are essentially identical to those exhibited by cells lacking Pmk1/Spm1, a recently identified mitogen-activated protein kinase. Our evidence suggests that Pmk1/Spm1 acts downstream from Mkh1 in a common pathway. Our results also suggest that Mkh1 and Pck2 act independently to maintain cell wall integrity, cell morphology, and salt resistance but act in opposition to regulate filamentous growth.

Mammalian CAP interacts with CAP, CAP2, and actin.

We previously identified human CAP, a homolog of the yeast adenylyl cyclase—associated protein. Previous studies suggest that the N‐terminal and C‐terminal domains of CAP have distinct functions. We have explored the interactions of human CAP with various proteins. First, by performing yeast two‐hybrid screens, we have identified peptides from several proteins that interact with the C‐terminal and/or the N‐terminal domains of human CAP. These peptides include regions derived from CAP and BAT3, a protein with unknown function. We have further shown that MBP fusions with these peptides can associate in vitro with the N‐terminal or C‐terminal domains of CAP fused to GST. Our observations indicate that CAP contains regions in both the N‐terminal and C‐terminal domains that are capable of interacting with each other or with themselves. Furthermore, we found that myc‐epitope‐tagged CAP coimmunoprecipitates with HA‐epitope‐tagged CAP from either yeast or mammalian cell extracts. Similar results demonstrate that human CAP can also interact with human CAP2. We also show that human CAP interacts with actin, both by the yeast two‐hybrid test and by coimmunoprecipitation of epitope‐tagged CAP from yeast or mammalian cell extracts. This interaction requires the C‐terminal domain of CAP, but not the N‐terminal domain. Thus CAP appears to be capable of interacting in vivo with other CAP molecules, CAP2, and actin. We also show that actin co‐immunoprecipitates with HA‐CAP2 from mammalian cell extracts.

Differential expression of CAP and CAP2 in adult rat tissues.

We previously reported the identification of the human CAP and CAP2 genes which encode proteins related to the yeast adenylyl cyclase (CYR)-associated CAP protein. The rat CAP homolog, MCH1, has also been previously cloned. We have cloned a cDNA encoding the rat homolog of CAP2. Rat CAP/MCH1 and CAP2 are 63% identical to each other. Using the reverse transcription-polymerase chain reaction (RT-PCR) method, we have examined CAP/MCH1 and CAP2 mRNA levels in various adult rat tissues. Our results show a dramatic difference in the pattern of expression of these two genes. Consistent with previous reports, we detected CAP/MCH1 mRNA in all tissues examined; however, levels vary substantially between tissues. In particular, we found that CAP/MCH1 mRNA are present at relatively high levels in spleen, testes and lung, at moderate levels in brain, kidney, liver and small intestine, and at significantly lower levels in heart, skeletal muscle and skin. We have also investigated the levels of CAP/MCH1 in rat tissues by immunoblotting with a polyclonal antibody raised against a human CAP::GST fusion protein. In general, we find that the CAP/MCH1 mRNA levels reflect the amount of CAP/MCH1 found in different tissues. In contrast, CAP2 transcripts were present at relatively high levels in testes, at moderate levels in brain, heart and skeletal muscle, at lower levels in lung, skin, kidney and small intestine, and were undetectable in liver or spleen. The differences between the sequences and expression patterns of CAP/MCH1 and CAP2 are significant and suggest that these proteins have distinct functional roles.

ING1a expression increases during replicative senescence and induces a senescent phenotype.

The ING family of tumor suppressor proteins affects cell growth, apoptosis and response to DNA damage by modulating chromatin structure through association with different HAT and HDAC complexes. The major splicing isoforms of the ING1 locus are ING1a and INGlb. While INGlb plays a role in inducing apoptosis, the function of ING1a is currently unknown. Here we show that alternative splicing of the ING1 message alters the INGla:INGlb ratio by ~30‐fold in senescent compared to low passage primary fibroblasts. INGla antagonizes INGlb function in apoptosis, induces the formation of structures resembling senescence‐associated heterochromatic foci containing heterochromatin protein 1 gamma, the accumulation of senescence‐associated β‐galactosidase activity and promotes senescent cell morphology and cell cycle arrest. Phenotypic effects may result from differential effects on gene expression since ING1a increases levels of both retinoblastoma and the p16 cyclin‐dependent kinase inhibitor and ING1a and ING1b have opposite effects on the expression of proliferating nuclear cell antigen (PCNA), which is required for cell growth. Gene expression appears to be altered by targeting of HDAC complexes to gene promoters since INGla associates with several‐fold higher levels of HDAC1 in senescent, compared to replication‐competent cells and ING1 is found on the PCNA promoter by chromatin immunoprecipitation analysis. These data demonstrate a novel role for the ING1 proteins in differentially regulating senescence‐associated chromatin remodeling vs. apoptosis and support the idea that altered ratios of the ING1 splicing isoforms may contribute to establishing the senescent phenotype through HDAC and HAT complex‐mediated effects on chromatin structure.

CIRCULAR YAC VECTORS CONTAINING A SMALL MAMMALIAN ORIGIN SEQUENCE CAN ASSOCIATE WITH THE NUCLEAR MATRIX

Three different mammalian origins of DNA replication, 343, S3, and X24, have been cloned into a 15.8 kb circular yeast vector pYACneo. Subsequent transfection into HeLa cells resulted in the isolation of several stably maintained clones. Two cell lines, C343e2 and CS3e1, were found to have sequences maintained as episomes in long‐term culture with a stability per generation of approximately 80%. Both episomes also contain matrix attachment region (MAR) sequences which mediate the binding of DNA to the nuclear skeleton and are thought to play a role in DNA replication. Using high salt extraction of the nucleus and fluorescent in situ hybridization, we were able to demonstrate an association of the 343 episome with the nuclear matrix, most probably through functional MAR sequences that allow an association with the nuclear matrix and associated regions containing essential replication proteins. The presence of functional MARs in small episomal sequences may facilitate the replication and maintenance of transfected DNA as an episome and improve their utility as small episomal constructs, potential microchromosomes. J. Cell. Biochem. 67:439–450, 1997.

Nak1 interacts with Hob1 and Wsp1 to regulate cell growth and polarity in Schizosaccharomyces pombe

We have previously reported that Nak1, a group-II germinal center (GC) kinase, is essential for polarized growth in Schizosaccharomyces pombe. Here, we provide evidence that Nak1 regulates cell growth and polarity, in part, through its interactions with Hob1 (an Rvs167/amphiphysin homolog) and Wsp1 (Wiskott-Aldrich-syndrome-protein homolog). We found that Nak1, Hob1 and Wsp1 interact physically, and that both Hob1/green-fluorescent-protein (Hob1-GFP) and Wsp1-GFP fusion proteins localized to F-actin patches at growing cell ends and medial division sites. Hob1-GFP was dissociated from patches in cells lacking Wsp1. Also, Hob1 overexpression dissociated Wsp1-GFP from foci, inhibited Wsp1-directed F-actin formation in vitro and partially restored polarity defects associated with Wsp1 overexpression or nak1 repression. Furthermore, loss of both Wsp1 and Hob1 resulted in rounded cells, slow growth and multiple septae. Together, these observations suggest that Hob1 and Wsp1 cooperate to mediate cell polarity, growth and division. Repression of nak1 resulted in a random redistribution of Hob1-GFP and Wsp1-GFP foci, and inhibition of Wsp1-directed F-actin formation in vitro. Furthermore, hob1Δ and wsp1Δ mutants exhibited synthetic growth defects in combination with nak1 repression, suggesting that Nak1 has redundant functions with Hob1 and Wsp1. Collectively, our results suggest that Nak1 both regulates and cooperates with Hob1 and Wsp1 to promote F-actin formation and polarized cell growth.

Raf60, a Novel Component of the Rpd3 Histone Deacetylase Complex Required for Rpd3 Activity in Saccharomyces cerevisiae

The Rpd3 histone deacetylase complex contains several previously characterized proteins, including Rpd3, Sin3, Sds3, Sap30, and Pho23. We purified the Rdp3 complex to near homogeneity using the tandem affinity purification method. Mass spectrometric analysis revealed the presence of a novel component, which we named Raf60. We showed that Myc-Raf60 co-fractionated with Rpd3-TAP by gel filtration chromatography, and both Myc-Rpd3 and Sin3 co-immunoprecipitated with HA-Raf60. In addition, HA-Raf60 immunoprecipitates displayed Rpd3-dependent histone deacetylase activity, and raf60 deletion resulted in loss of Rpd3 complex activity, as measured by in vitro assays. Furthermore, we found that raf60Δ cells exhibited phenotypes similar to those of rpd3Δ cells, including derepression of secreted acid phosphatase (Pho5), hypersensitivity to cycloheximide, and hypersensitivity to heat shock. Also, we found by reverse transcription-PCR that raf60Δ cells, similar to rpd3Δ cells, displayed elevated levels of PHO5 and INO1 mRNA. Our results demonstrate that Raf60 is a component of the Rpd3 histone deacetylase complex and that it is required for normal Rpd3 complex activity and repression of gene expression.