Functional Separation of the Requirements for Establishment and Maintenance of Centromeric Heterochromatin

Abstract

The establishment and maintenance of centromeric heterochromatin in fission yeast require the RITS complex. Comprised of centromeric siRNAs, the chromodomain protein Chp1, Argonaute (Ago1), and Tas3, RITS couples the cellular RNAi pathway with assembly of constitutive heterochromatin. However, the mechanisms governing RITS-dependent establishment versus maintenance of centromeric heterochromatin remain unresolved. Here, we report that a mutant Tas3 protein that cannot bind Ago1 supports the maintenance of centromeric heterochromatin but cannot mediate efficient de novo establishment from cells transiently depleted for the histone H3 lysine 9 methyltransferase Clr4. In contrast, centromeric heterochromatin efficiently assembles in mutant cells transiently depleted for dicer. This mutant therefore allows ordering of the events leading to establishment of centromeric heterochromatin and places lysine 9 methylation of histone H3 upstream of dicer function.

Continuous Requirement for the Clr4 Complex But Not RNAi for Centromeric Heterochromatin Assembly in Fission Yeast Harboring a Disrupted RITS Complex

“…In addition, dcr1Δ cells cannot generate siRNAs from centromeric transcripts, but on reintegration of dcr1 + , siRNA production resumed efficiently in both tas3-TAP and tas3 WG - TAP backgrounds (Figure S2D). Together, these results confirmed and extended our data obtained with overexpressed dcr1 + [10]. dcr1 + and siRNAs are not critical for Clr-C activity at centromeres, but are important for amplification of the H3K9me2 signal during later stages of heterochromatin assembly.…”

Continuous Requirement for the Clr4 Complex But Not RNAi for Centromeric Heterochromatin Assembly in Fission Yeast Harboring a Disrupted RITS Complex

“…In addition, dcr1Δ cells cannot generate siRNAs from centromeric transcripts, but on reintegration of dcr1 + , siRNA production resumed efficiently in both tas3-TAP and tas3 WG - TAP backgrounds (Figure S2D). Together, these results confirmed and extended our data obtained with overexpressed dcr1 + [10]. dcr1 + and siRNAs are not critical for Clr-C activity at centromeres, but are important for amplification of the H3K9me2 signal during later stages of heterochromatin assembly.…”

Sir2 is required for Clr4 to initiate centromeric heterochromatin assembly in fission yeast

“…Indeed, we present here that Sir2 and Clr3 play redundant roles in the maintenance of centromeric heterochromatin (Figure 3), as has recently been reported (Buscaino et al 2013). Additionally, we demonstrate an important dependence on Sir2 for Clr4 function during the RNAi-independent establishment of heterochromatin at centromeres (Figure 7; Partridge et al, 2007;Shanker et al, 2010). We note that Sir2 enzymatic activity has recently been reported as sufficient and necessary for maintenance of heterochromatin in RNAi-deficient yeast (Buscaino et al, 2013), although this study did not reveal a dependence on Sir2 for heterochromatin initiation.…”

Epigenetic Regulation of Pericentromeric Heterochromatin during Mammalian Meiosis

“…It has previously been shown that methylation of H3K9 by Clr4, a histone methyltransferase homologous to SUV39H1 in mammals, acts upstream of DICER in the establishment of centromeric heterochromatin in fission yeast [Partridge et al, 2007]. Our observations that H3K9me2 is enriched on heterochromatin during meiosis prior to DICER and RNA polymerase II further support this finding in yeast and suggest that the order of events leading to heterochromatin formation may be conserved between yeast and mammals.…”

Continuous Requirement for the Clr4 Complex But Not RNAi for Centromeric Heterochromatin Assembly in Fission Yeast Harboring a Disrupted RITS Complex

“…In addition, dcr1Δ cells cannot generate siRNAs from centromeric transcripts, but on reintegration of dcr1 + , siRNA production resumed efficiently in both tas3-TAP and tas3 WG - TAP backgrounds (Figure S2D). Together, these results confirmed and extended our data obtained with overexpressed dcr1 + [10]. dcr1 + and siRNAs are not critical for Clr-C activity at centromeres, but are important for amplification of the H3K9me2 signal during later stages of heterochromatin assembly.…”

Sir2 is required for Clr4 to initiate centromeric heterochromatin assembly in fission yeast

“…Indeed, we present here that Sir2 and Clr3 play redundant roles in the maintenance of centromeric heterochromatin (Figure 3), as has recently been reported (Buscaino et al 2013). Additionally, we demonstrate an important dependence on Sir2 for Clr4 function during the RNAi-independent establishment of heterochromatin at centromeres (Figure 7; Partridge et al, 2007;Shanker et al, 2010). We note that Sir2 enzymatic activity has recently been reported as sufficient and necessary for maintenance of heterochromatin in RNAi-deficient yeast (Buscaino et al, 2013), although this study did not reveal a dependence on Sir2 for heterochromatin initiation.…”

Epigenetic Regulation of Pericentromeric Heterochromatin during Mammalian Meiosis

“…It has previously been shown that methylation of H3K9 by Clr4, a histone methyltransferase homologous to SUV39H1 in mammals, acts upstream of DICER in the establishment of centromeric heterochromatin in fission yeast [Partridge et al, 2007]. Our observations that H3K9me2 is enriched on heterochromatin during meiosis prior to DICER and RNA polymerase II further support this finding in yeast and suggest that the order of events leading to heterochromatin formation may be conserved between yeast and mammals.…”

Continuous Requirement for the Clr4 Complex But Not RNAi for Centromeric Heterochromatin Assembly in Fission Yeast Harboring a Disrupted RITS Complex

“…In addition, dcr1Δ cells cannot generate siRNAs from centromeric transcripts, but on reintegration of dcr1 + , siRNA production resumed efficiently in both tas3-TAP and tas3 WG - TAP backgrounds (Figure S2D). Together, these results confirmed and extended our data obtained with overexpressed dcr1 + [10]. dcr1 + and siRNAs are not critical for Clr-C activity at centromeres, but are important for amplification of the H3K9me2 signal during later stages of heterochromatin assembly.…”

Sir2 is required for Clr4 to initiate centromeric heterochromatin assembly in fission yeast

“…Indeed, we present here that Sir2 and Clr3 play redundant roles in the maintenance of centromeric heterochromatin (Figure 3), as has recently been reported (Buscaino et al 2013). Additionally, we demonstrate an important dependence on Sir2 for Clr4 function during the RNAi-independent establishment of heterochromatin at centromeres (Figure 7; Partridge et al, 2007;Shanker et al, 2010). We note that Sir2 enzymatic activity has recently been reported as sufficient and necessary for maintenance of heterochromatin in RNAi-deficient yeast (Buscaino et al, 2013), although this study did not reveal a dependence on Sir2 for heterochromatin initiation.…”

Epigenetic Regulation of Pericentromeric Heterochromatin during Mammalian Meiosis

“…It has previously been shown that methylation of H3K9 by Clr4, a histone methyltransferase homologous to SUV39H1 in mammals, acts upstream of DICER in the establishment of centromeric heterochromatin in fission yeast [Partridge et al, 2007]. Our observations that H3K9me2 is enriched on heterochromatin during meiosis prior to DICER and RNA polymerase II further support this finding in yeast and suggest that the order of events leading to heterochromatin formation may be conserved between yeast and mammals.…”