Biomedical subjects

Hiten D Madhani

Publications and source records attributed to Hiten D Madhani.

13 recordsLinked to original sources

PubMed2025

CRISPR/Cas9-compatible plasmids enabling seven dominant genetic selection methods for the human fungal pathogen Cryptococcus neoformans.

Cryptococcus neoformans is the most common cause of human fungal meningitis and an important model system for studying fundamental eukaryotic biology. Genetic manipulation of this organism relies on three dominant drug resistance markers (nourseothricin acetyltransferase [NAT], neomycin phosphotransferase II [NEO], and hygromycin B phosphotransferase [HYG]) and the recyclable dominant prototrophic marker amdS. With ongoing technological advances that are expanding our ability to explore cryptococcal gene function, contemporary studies often require multiple genetic manipulations in the same strain. Additional dominant selection methods would maximize the utility of these tools by facilitating their combinatorial use. Here, we identify blasticidin S resistance via the blasticidin S deaminase (BSD) or blasticidin S resistance (BSR) markers as a novel dominant selection method for C. neoformans. We further validate phleomycin resistance via the bleomycin resistance gene (BLE) marker as an additional selection method, confirming a study that first established this marker 25 years ago (J. Hua, J. D. Meyer, and J. K. Lodge, Clin Diagn Lab Immunol 7:125-128, 2000, https://doi.org/10.1128/cdli.7.1.125-128.2000). To enable highly efficient CRISPR/Cas9-mediated genome modification, we incorporated these markers, as well as the newly established dominant prototrophic marker ptxD (M. Khongthongdam, T. Phetruen, and S. Chanarat, Microbiol Spectr 13:e01618-24, 2025, https://doi.org/10.1128/spectrum.01618-24), into a vector series that enables the construction of fused marker-sgRNA products via PCR. Altogether, this work expands the number of dominant genetic selection methods for C. neoformans to seven, including five drug selection regimes and two prototrophic methods. The vector series has been deposited at Addgene. IMPORTANCE Cryptococcus neoformans is the top-ranked World Health Organization priority fungal pathogen due to its widespread distribution and inadequate treatment options. Additionally, as a basidiomycete yeast occupying an underexplored branch of the fungal kingdom, this organism is a powerful system for deciphering core eukaryotic biology that is absent in classic model fungi. Defining functions for novel cryptococcal genes is a crucial priority, and the availability of additional genetic selection methods would facilitate these efforts. In this study, we establish blasticidin S resistance as a novel genetic selection method for C. neoformans, and we validate a previous report using phleomycin resistance as such. This work expands the number of reliable dominant selection methods to seven, providing flexibility for the introduction of sequential genetic modifications into single strains.

Cryptococcus neoformans↗

PubMed2006

Histone H3 lysine 36 methylation antagonizes silencing in Saccharomyces cerevisiae independently of the Rpd3S histone deacetylase complex.

In yeast, methylation of histone H3 on lysine 36 (H3-K36) is catalyzed by the NSD1 leukemia oncoprotein homolog Set2. The histone deacetylase complex Rpd3S is recruited to chromatin via binding of the chromodomain protein Eaf3 to methylated H3-K36 to prevent erroneous transcription initiation. Here we identify a distinct function for H3-K36 methylation. We used random mutagenesis of histones H3 and H4 followed by a reporter-based screen to identify residues necessary to prevent the ectopic spread of silencing from the silent mating-type locus HMRa into flanking euchromatin. Mutations in H3-K36 or deletion of SET2 caused ectopic silencing of a heterochromatin-adjacent reporter. Transcriptional profiling revealed that telomere-proximal genes are enriched for those that display decreased expression in a set2Delta strain. Deletion of SIR4 rescued the expression defect of 26 of 37 telomere-proximal genes with reduced expression in set2Delta cells, implying that H3-K36 methylation prevents the spread of telomeric silencing. Indeed, Sir3 spreads from heterochromatin into neighboring euchromatin in set2Delta cells. Furthermore, genetic experiments demonstrated that cells lacking the Rpd3S-specific subunits Eaf3 or Rco1 did not display the anti-silencing phenotype of mutations in SET2 or H3-K36. Thus, antagonism of silencing is independent of the only known effector of this conserved histone modification.

Euchromatin↗

PubMed2006

Patterning chromatin: form and function for H2A.Z variant nucleosomes.

Although many histone variants are specific to higher eukaryotes, the H2A variant H2A.Z has been conserved during eukaryotic evolution. Genetic studies have demonstrated roles for H2A.Z in antagonizing gene-silencing, chromosome stability and gene activation. Biochemical work has identified a conserved chromatin-remodeling complex responsible for H2A.Z deposition. Recent studies have shown that two H2A.Z nucleosomes flank a nucleosome-free region containing the transcription initiation site in promoters of both active and inactive genes in Saccharomyces cerevisiae. This chromatin pattern is generated through the action of a DNA deposition signal and a specific pattern of histone tail acetylation.

Animals↗

PubMed2006

Control of MAPK signaling specificity by a conserved residue in the MEK-binding domain of the yeast scaffold protein Ste5.

The yeast kinase scaffold Ste5 has been proposed to prevent unwanted cross-talk between the pheromone response pathway and other MAPK cascades. Protein fusion experiments have demonstrated that covalently tethering signaling components to each other or to Ste5 can determine the outcome of signaling. However, these do not fully test the role of scaffolds in signaling specificity, since fusing components precludes differential dissociation of subpopulations. We performed a targeted genetic screen on STE5 and repeatedly identified recessive mutations in a conserved residue, E756, in the Ste7/MEK-binding domain that caused erroneous activation of the filamentation MAPK pathway by pheromone signaling. Mutant cells exhibited a shift in the MAPK activation pattern such that the filamentation MAPK Kss1 was predominately activated in response to pheromone. Velocity sedimentation studies showed that the mutant scaffold was defective in binding to a phosphorylated subpopulation of Ste7. Our data suggest that increased dissociation of activated Ste7 kinase from the mutant scaffold may cause the observed shift in MAPK activation from Fus3 to Kss1 and the resulting loss of specificity. Cross-talk in ste5-E756G cells was due to both increased activation of Kss1 and reduced Fus3-dependent degradation of the filamentation pathway transcription factor Tec1. These studies demonstrate a role for an endogenous scaffold in signaling specificity.

Adaptor Proteins, Signal Transducing↗

PubMed2006

Selective kinase inhibition by exploiting differential pathway sensitivity.

Protein kinase inhibitors are optimized to have high affinity for their intended target(s) to elicit the desired cellular effects. Here, we asked whether differences in inhibitory sensitivity between two kinase signaling pathways, controlled by the cyclin-dependent kinases Cdk1 and Pho85, can be sufficient to allow for selective targeting of one pathway over the other. We show the oxindole inhibitor GW297361 elicits a Pho85-selective response in cells despite having a 20-fold greater biochemical potency for Cdk1 in vitro. We provide evidence that partial inhibition of Pho85 is sufficient to activate Pho85-dependent signaling, but partial inhibition of Cdk1 is not sufficient to block Cdk1-dependent cell proliferation. Identification of highly sensitive kinases may provide a means to achieve selective perturbation of kinase signaling pathways complementary to efforts to achieve maximal differences between in vitro IC50 values.

Base Sequence↗

PubMed2006

Functional analysis of protein kinase networks in living cells: beyond "knock-outs" and "knock-downs".

The identification of over 500 protein kinases encoded by the human genome sequence offers one measure of the importance of protein kinase networks in cell biology. High throughput technologies for inactivating genes are producing an awe-inspiring amount of data on the cellular and organismal effects of reducing the levels of individual protein kinases. Despite these technical advances, our understanding of kinase networks remains imprecise. Major challenges include correctly assigning kinases to particular networks, understanding how they are regulated, and identifying the relevant in vivo substrates. Genetic methods provide a way of addressing these questions, but their application requires understanding the nuances of how different types of mutations can affect protein kinases. The goal of this article is to provide a brief introductory primer into these issues using examples from yeast MAPK cascades and to motivate future systematic genetic analysis focusing on individual residues of protein kinases.

Genetic Techniques↗

PubMed2005

Histone variant H2A.Z marks the 5' ends of both active and inactive genes in euchromatin.

In S. cerevisiae, histone variant H2A.Z is deposited in euchromatin at the flanks of silent heterochromatin to prevent its ectopic spread. We show that H2A.Z nucleosomes are found at promoter regions of nearly all genes in euchromatin. They generally occur as two positioned nucleosomes that flank a nucleosome-free region (NFR) that contains the transcription start site. Astonishingly, enrichment at 5' ends is observed not only at actively transcribed genes but also at inactive loci. Mutagenesis of a typical promoter revealed a 22 bp segment of DNA sufficient to program formation of a NFR flanked by two H2A.Z nucleosomes. This segment contains a binding site of the Myb-related protein Reb1 and an adjacent dT:dA tract. Efficient deposition of H2A.Z is further promoted by a specific pattern of histone H3 and H4 tail acetylation and the bromodomain protein Bdf1, a component of the Swr1 remodeling complex that deposits H2A.Z.

Acetylation↗

PubMed2005

Chemical genomic profiling to identify intracellular targets of a multiplex kinase inhibitor.

The identification of the kinase or kinases targeted by protein kinase inhibitors is a critical challenge in validating their use as therapeutic agents or molecular probes. Here, to address this problem, we describe a chemical genomics strategy that uses a direct comparison between microarray transcriptional signatures elicited by an inhibitor of unknown specificity and those elicited by highly specific pharmacological inhibition of engineered candidate kinase targets. By using this approach, we have identified two cyclin-dependent kinases, Cdk1 and Pho85, as the targets of the inhibitor GW400426 in Saccharomyces cerevisiae. We demonstrate that simultaneous inhibition of Cdk1 and Pho85, and not inhibition of either kinase alone, by GW400426 controls the expression of specific transcripts involved in polarized cell growth, thus revealing a cellular process that is uniquely sensitive to the multiplex inhibition of these two kinases. Our results suggest that the cellular responses induced by multiplex protein kinase inhibitors may be an emergent property that cannot be understood fully by considering only the sum of individual inhibitor-kinase interactions.

CDC2 Protein Kinase↗

PubMed2004

Pheromone-dependent destruction of the Tec1 transcription factor is required for MAP kinase signaling specificity in yeast.

The yeast MAPK pathways required for mating versus filamentous growth share multiple components yet specify distinct programs. The mating-specific MAPK, Fus3, prevents crosstalk between the two pathways by unknown mechanisms. Here we show that pheromone signaling induces Fus3-dependent degradation of Tec1, the transcription factor specific to the filamentation pathway. Degradation requires Fus3 kinase activity and a MAPK phosphorylation site in Tec1 at threonine 273. Fus3 associates with Tec1 in unstimulated cells, and active Fus3 phosphorylates Tec1 on T273 in vitro. Destruction of Tec1 requires the F box protein Dia2 (Digs-into-agar-2), and Cdc53, the Cullin of SCF (Skp1-Cdc53-F box) ubiquitin ligases. Notably, mutation of the phosphoacceptor site in Tec1, deletion of FUS3, or deletion of DIA2 results in a loss of signaling specificity such that pheromone pathway signaling erroneously activates filamentation pathway gene expression and invasive growth. Signal-induced destruction of a transcription factor for a competing pathway provides a mechanism for signaling specificity.

Amino Acid Sequence↗

PubMed2004

A protein complex containing the conserved Swi2/Snf2-related ATPase Swr1p deposits histone variant H2A.Z into euchromatin.

The conserved histone variant H2A.Z functions in euchromatin to antagonize the spread of heterochromatin. The mechanism by which histone H2A is replaced by H2A.Z in the nucleosome is unknown. We identified a complex containing 13 different polypeptides associated with a soluble pool of H2A.Z in Saccharomyces cerevisiae. This complex was designated SWR1-Com in reference to the Swr1p subunit, a Swi2/Snf2-paralog. Swr1p and six other subunits were found only in SWR1-Com, whereas six other subunits were also found in the NuA4 histone acetyltransferase and/or the Ino80 chromatin remodeling complex. H2A.Z and SWR1 were essential for viability of cells lacking the EAF1 component of NuA4, pointing to a close functional connection between these two complexes. Strikingly, chromatin immunoprecipitation analysis of cells lacking Swr1p, the presumed ATPase of the complex, revealed a profound defect in the deposition of H2A.Z at euchromatic regions that flank the silent mating type cassette HMR and at 12 other chromosomal sites tested. Consistent with a specialized role for Swr1p in H2A.Z deposition, the majority of the genome-wide transcriptional defects seen in swr1Delta cells were also found in htz1Delta cells. These studies revealed a novel role for a member of the ATP-dependent chromatin remodeling enzyme family in determining the region-specific histone subunit composition of chromatin in vivo and controlling the epigenetic state of chromatin. Metazoan orthologs of Swr1p (Drosophila Domino; human SRCAP and p400) may have analogous functions.

Adenosine Triphosphatases↗

PubMed2004

Principles of MAP kinase signaling specificity in Saccharomyces cerevisiae.

Cells respond to a plethora of signals using a limited set of intracellular signal transduction components. Surprisingly, pathways that transduce distinct signals can share protein components, yet avoid erroneous cross-talk. A highly tractable model system in which to study this paradox is the yeast Saccharomyces cerevisiae, which harbors three mitogen-activated protein kinase (MAPK) signal transduction cascades that share multiple signaling components. In this review we first describe potential mechanisms by which specificity could be achieved by signaling pathways that share components. Second, we summarize key features and components of the yeast MAPK pathways that control the mating pheromone response, filamentous growth, and the response to high osmolarity. Finally, we review biochemical analyses in yeast of mutations that cause cross-talk between these three MAPK pathways and their implications for the mechanistic bases for signaling specificity. Although much remains to be learned, current data indicate that scaffolding and cross pathway inhibition play key roles in the maintenance of fidelity.

Gene Expression Regulation, Fungal↗

PubMed2003

Conserved histone variant H2A.Z protects euchromatin from the ectopic spread of silent heterochromatin.

Boundary elements hinder the spread of heterochromatin, yet these sites do not fully account for the preservation of adjacent euchromatin. Histone variant H2A.Z (Htz1 in yeast) replaces conventional H2A in many nucleosomes. Microarray analysis revealed that HTZ1-activated genes cluster near telomeres. The reduced expression of most of these genes in htz1Delta cells was reversed by the deletion of SIR2 (sir2Delta) suggesting that H2A.Z antagonizes telomeric silencing. Other Htz1-activated genes flank the silent HMR mating-type locus. Their requirement for Htz1 can be bypassed by sir2Delta or by a deletion encompassing the silencing nucleation sites in HMR. In htz1Delta cells, Sir2 and Sir3 spread into flanking euchromatic regions, producing changes in histone H4 acetylation and H3 4-methylation indicative of ectopic heterochromatin formation. Htz1 is enriched in these euchromatic regions and acts synergistically with a boundary element to prevent the spread of heterochromatin. Thus, euchromatin and heterochromatin each contains components that antagonize switching to the opposite chromatin state.

Cell Nucleus↗

PubMed2003

A conserved RING finger protein required for histone H2B monoubiquitination and cell size control.

Monoubiquitination of histone H2B is required for methylation of histone H3 on lysine 4 (K4), a modification associated with active chromatin. The identity of the cognate ubiquitin ligase is unknown. We identify Bre1 as an evolutionarily conserved RING finger protein required in vivo for both H2B ubiquitination and H3 K4 methylation. The RING domain of Bre1 is essential for both of these modifications as is Lge1 (Large 1), a protein required for cell size control that copurifies with Bre1. In cells lacking the euchromatin-associated histone variant H2A.Z, BRE1, RAD6, and LGE1 are each essential for cell viability, supporting redundant functions for H2B ubiquitination and H2A substitution in the formation of active chromatin. Notably, analysis of mutants demonstrates a function for Bre1/Lge1-dependent H2B monoubiquitination in the control of cell size.

Amino Acid Sequence↗