Chromatin Immunoprecipitation (ChIP) Assay in Candida albicans

Mitra, S.; Shanker, Laxmi; Chatterjee, Gautam; Sanyal, Kaustuv

doi:10.1007/978-1-4939-3052-4_4

Cited by 7 publications

(5 citation statements)

References 22 publications

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“…Having identified authentic kinetochore proteins, we next sought to map the centromeres in the C . tropicalis genome as the binding sites of CENP-A and CENP-C by chromatin immunoprecipitation followed by next generation sequencing (ChIP-seq) [ 48 ]. The sequenced C .…”

Section: Resultsmentioning

confidence: 99%

Repeat-Associated Fission Yeast-Like Regional Centromeres in the Ascomycetous Budding Yeast Candida tropicalis

et al. 2016

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The centromere, on which kinetochore proteins assemble, ensures precise chromosome segregation. Centromeres are largely specified by the histone H3 variant CENP-A (also known as Cse4 in yeasts). Structurally, centromere DNA sequences are highly diverse in nature. However, the evolutionary consequence of these structural diversities on de novo CENP-A chromatin formation remains elusive. Here, we report the identification of centromeres, as the binding sites of four evolutionarily conserved kinetochore proteins, in the human pathogenic budding yeast Candida tropicalis. Each of the seven centromeres comprises a 2 to 5 kb non-repetitive mid core flanked by 2 to 5 kb inverted repeats. The repeat-associated centromeres of C. tropicalis all share a high degree of sequence conservation with each other and are strikingly diverged from the unique and mostly non-repetitive centromeres of related Candida species—Candida albicans, Candida dubliniensis, and Candida lusitaniae. Using a plasmid-based assay, we further demonstrate that pericentric inverted repeats and the underlying DNA sequence provide a structural determinant in CENP-A recruitment in C. tropicalis, as opposed to epigenetically regulated CENP-A loading at centromeres in C. albicans. Thus, the centromere structure and its influence on de novo CENP-A recruitment has been significantly rewired in closely related Candida species. Strikingly, the centromere structural properties along with role of pericentric repeats in de novo CENP-A loading in C. tropicalis are more reminiscent to those of the distantly related fission yeast Schizosaccharomyces pombe. Taken together, we demonstrate, for the first time, fission yeast-like repeat-associated centromeres in an ascomycetous budding yeast.

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Section: Resultsmentioning

confidence: 99%

Repeat-Associated Fission Yeast-Like Regional Centromeres in the Ascomycetous Budding Yeast Candida tropicalis

et al. 2016

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“…The ChIP assays were performed as described previously by Chatterjee and colleagues and Mitra and colleagues [56, 58]. Briefly, each strain was grown until exponential phase (approximately 2 × 10 7 cells/ml) or grown in biofilm condition for 24 hours, and cells were cross-linked with 1% final concentration of formaldehyde for 13 minutes.…”

Section: Methodsmentioning

confidence: 99%

The Candida albicans biofilm gene circuit modulated at the chromatin level by a recent molecular histone innovation

et al. 2019

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Histone H3 and its variants regulate gene expression but the latter are absent in most ascomycetous fungi. Here, we report the identification of a variant histone H3, which we have designated H3V CTG because of its exclusive presence in the CTG clade of ascomycetes, including Candida albicans , a human pathogen. C . albicans grows both as single yeast cells and hyphal filaments in the planktonic mode of growth. It also forms a three-dimensional biofilm structure in the host as well as on human catheter materials under suitable conditions. H3V CTG null ( hht1/hht1 ) cells of C . albicans are viable but produce more robust biofilms than wild-type cells in both in vitro and in vivo conditions. Indeed, a comparative transcriptome analysis of planktonic and biofilm cells reveals that the biofilm circuitry is significantly altered in H3V CTG null cells. H3V CTG binds more efficiently to the promoters of many biofilm-related genes in the planktonic cells than during biofilm growth, whereas the binding of the core canonical histone H3 on the corresponding promoters largely remains unchanged. Furthermore, biofilm defects associated with master regulators, namely, biofilm and cell wall regulator 1 (Bcr1), transposon enhancement control 1 (Tec1), and non-dityrosine 80 (Ndt80), are significantly rescued in cells lacking H3V CTG . The occupancy of the transcription factor Bcr1 at its cognate promoter binding sites was found to be enhanced in the absence of H3V CTG in the planktonic form of growth resulting in enhanced transcription of biofilm-specific genes. Further, we demonstrate that co-occurrence of valine and serine at the 31st and 32nd positions in H3V CTG , respectively, is essential for its function. Taken together, we show that even in a unicellular organism, differential gene expression patterns are modulated by the relative occupancy of the specific histone H3 type at the chromatin level.

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“…The ChIP assays were performed as described previously (Mitra et al, 2016). Briefly, each strain was grown until the exponential phase (approximately 2 × 10 7 cells/mL), and cells were cross‐linked with a 1% final concentration of formaldehyde for 15 minutes.…”

Section: Methodsmentioning

confidence: 99%

Negative regulation of biofilm development by the CUG‐Ser1 clade‐specific histone H3 variant is dependent on the canonical histone chaperone CAF‐1 complex in Candida albicans

Singha

Aggarwal

Sanyal

2023

Molecular Microbiology

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The CUG‐Ser1 clade‐specific histone H3 variant (H3VCTG) has been reported to be a negative regulator of planktonic to biofilm growth transition in Candida albicans. The preferential binding of H3VCTG at the biofilm gene promoters makes chromatin repressive for the biofilm mode of growth. The two evolutionarily conserved chaperone complexes involved in incorporating histone H3 are CAF‐1 and HIRA. In this study, we sought to identify the chaperone complex(es) involved in loading H3VCTG. We demonstrate that C. albicans cells lacking either Cac1 or Cac2 subunit of the CAF‐1 chaperone complex, exhibit a hyper‐filamentation phenotype on solid surfaces and form more robust biofilms than wild‐type cells, thereby mimicking the phenotype of the H3VCTG null mutant. None of the subunits of the HIRA chaperone complex shows any significant difference in biofilm growth as compared to the wild type. The occupancy of H3VCTG is found to be significantly reduced at the promoters of biofilm genes in the absence of CAF‐1 subunits. Hence, we provide evidence that CAF‐1, a chaperone known to load canonical histone H3 in mammalian cells, is involved in chaperoning of variant histone H3VCTG at the biofilm gene promoters in C. albicans. Our findings also illustrate the acquisition of an unconventional role of the CAF‐1 chaperone complex in morphogenesis in C. albicans.

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Chromatin Immunoprecipitation (ChIP) Assay in Candida albicans

Cited by 7 publications

References 22 publications

Repeat-Associated Fission Yeast-Like Regional Centromeres in the Ascomycetous Budding Yeast Candida tropicalis

Repeat-Associated Fission Yeast-Like Regional Centromeres in the Ascomycetous Budding Yeast Candida tropicalis

The Candida albicans biofilm gene circuit modulated at the chromatin level by a recent molecular histone innovation

Negative regulation of biofilm development by the CUG‐Ser1 clade‐specific histone H3 variant is dependent on the canonical histone chaperone CAF‐1 complex in Candida albicans

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