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DNA methylation and transcription in a distal region upstream from the bovine AlphaS1 casein gene after once or twice daily milking.

Nguyen M, Boutinaud M, Pétridou B, Gabory A, Pannetier M, Chat S, Bouet S, Jouneau L, Jaffrezic F, Laloë D, Klopp C, Brun N, Kress C, Jammes H, Charlier M, Devinoy E - PLoS ONE (2014)

Bottom Line: Once daily milking (ODM) induces a reduction in milk production when compared to twice daily milking (TDM).Our results therefore shed light on mechanisms that help to explain how milk production is almost, but not fully, restored when TDM is resumed (15.1 ± 0.2 kg/day instead of 16.2 ± 0.2 kg/day, p<0.01).We now need to clarify whether the transcription of this novel RNA is a consequence of STAT5 interacting with the CSN1S1 distal region, or whether it plays a role in the chromatin structure of this region.

View Article: PubMed Central - PubMed

Affiliation: INRA, UMR1313 Génétique Animale et Biologie Intégrative, Jouy-en-Josas, France.

ABSTRACT
Once daily milking (ODM) induces a reduction in milk production when compared to twice daily milking (TDM). Unilateral ODM of one udder half and TDM of the other half, enables the study of underlying mechanisms independently of inter-individual variability (same genetic background) and of environmental factors. Our results show that in first-calf heifers three CpG, located 10 kb upstream from the CSN1S1 gene were methylated to 33, 34 and 28%, respectively, after TDM but these levels were higher after ODM, 38, 38 and 33%, respectively. These methylation levels were much lower than those observed in the mammary gland during pregnancy (57, 59 and 50%, respectively) or in the liver (74, 78 and 61%, respectively). The methylation level of a fourth CpG (CpG4), located close by (29% during TDM) was not altered after ODM. CpG4 methylation reached 39.7% and 59.5%, during pregnancy or in the liver, respectively. CpG4 is located within a weak STAT5 binding element, arranged in tandem with a second high affinity STAT5 element. STAT5 binding is only marginally modulated by CpG4 methylation, but it may be altered by the methylation levels of the three other CpG nearby. Our results therefore shed light on mechanisms that help to explain how milk production is almost, but not fully, restored when TDM is resumed (15.1 ± 0.2 kg/day instead of 16.2 ± 0.2 kg/day, p<0.01). The STAT5 elements are 100 bp away from a region transcribed in the antisense orientation, in the mammary gland during lactation, but not during pregnancy or in other reproductive organs (ovary or testes). We now need to clarify whether the transcription of this novel RNA is a consequence of STAT5 interacting with the CSN1S1 distal region, or whether it plays a role in the chromatin structure of this region.

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Binding activity of two potential STAT5 binding sites located within the CSN1S1 distal upstream region.(A) Representative EMSA. Mammary nuclear extract (22µg) from rabbits in early lactation were incubated with the indicated labelled 32P probes in the presence or absence of STAT5a or STAT5b antibodies (1 or 0.5 µg, respectively). Complexes were analysed on non denaturating 5% acrylamide gels in 0.25xTBE. The lower panel is an overexposed image of the upper one to show the weak interaction between D1 and nuclear extracts and the supershifts with STAT5 antibodies. (B) Competition assays with increasing amounts of unlabelled C, D2, D1 and D1m using 32P-C or 32P-D2, are indicated in each graph. Results are expressed as a percentage of the radioactivity bound in the absence of unlabelled oligonucleotides and plotted as a function of the log (ng oligonucleotide) in the reaction.
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pone-0111556-g006: Binding activity of two potential STAT5 binding sites located within the CSN1S1 distal upstream region.(A) Representative EMSA. Mammary nuclear extract (22µg) from rabbits in early lactation were incubated with the indicated labelled 32P probes in the presence or absence of STAT5a or STAT5b antibodies (1 or 0.5 µg, respectively). Complexes were analysed on non denaturating 5% acrylamide gels in 0.25xTBE. The lower panel is an overexposed image of the upper one to show the weak interaction between D1 and nuclear extracts and the supershifts with STAT5 antibodies. (B) Competition assays with increasing amounts of unlabelled C, D2, D1 and D1m using 32P-C or 32P-D2, are indicated in each graph. Results are expressed as a percentage of the radioactivity bound in the absence of unlabelled oligonucleotides and plotted as a function of the log (ng oligonucleotide) in the reaction.

Mentions: Mammary gland nuclear extracts incubated with the 32P labelled double strand D2 probe (32P-D2, corresponding to the more distal potential STAT5 sequence), formed a complex that migrated at a position similar to that observed for the Consensus STAT5 probe, indicated as C in Figure 1B (Figure 6A). The intensity of the signals detected for both complexes was similar. Over 90% of both D2 and C complexes were supershifted with STAT5a and STAT5b antibodies, clearly indicating that these potential STAT5 binding sites interact with both the STAT5a and STAT5b present in the extract. Weaker signals of the same mobility were also observed with 32P-D1. They were partially supershifted (15%) by STAT5 antibodies (Figure 6A, lower panel). The signals for 32P-D1m were even weaker (Figure 6A). Increasing the amounts of nuclear extracts did not increase the intensity of signals observed with D1 or D1m complexes, thus demonstrating that the weak band shifts were observed under saturating conditions (data not shown). These weak band shift signals could be anticipated by reported analysis of STAT5-DNA binding specificity [35]. The methylation of the CpG within the D1 sequence at most only marginally interferes with this weak in vitro interaction.


DNA methylation and transcription in a distal region upstream from the bovine AlphaS1 casein gene after once or twice daily milking.

Nguyen M, Boutinaud M, Pétridou B, Gabory A, Pannetier M, Chat S, Bouet S, Jouneau L, Jaffrezic F, Laloë D, Klopp C, Brun N, Kress C, Jammes H, Charlier M, Devinoy E - PLoS ONE (2014)

Binding activity of two potential STAT5 binding sites located within the CSN1S1 distal upstream region.(A) Representative EMSA. Mammary nuclear extract (22µg) from rabbits in early lactation were incubated with the indicated labelled 32P probes in the presence or absence of STAT5a or STAT5b antibodies (1 or 0.5 µg, respectively). Complexes were analysed on non denaturating 5% acrylamide gels in 0.25xTBE. The lower panel is an overexposed image of the upper one to show the weak interaction between D1 and nuclear extracts and the supershifts with STAT5 antibodies. (B) Competition assays with increasing amounts of unlabelled C, D2, D1 and D1m using 32P-C or 32P-D2, are indicated in each graph. Results are expressed as a percentage of the radioactivity bound in the absence of unlabelled oligonucleotides and plotted as a function of the log (ng oligonucleotide) in the reaction.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4219721&req=5

pone-0111556-g006: Binding activity of two potential STAT5 binding sites located within the CSN1S1 distal upstream region.(A) Representative EMSA. Mammary nuclear extract (22µg) from rabbits in early lactation were incubated with the indicated labelled 32P probes in the presence or absence of STAT5a or STAT5b antibodies (1 or 0.5 µg, respectively). Complexes were analysed on non denaturating 5% acrylamide gels in 0.25xTBE. The lower panel is an overexposed image of the upper one to show the weak interaction between D1 and nuclear extracts and the supershifts with STAT5 antibodies. (B) Competition assays with increasing amounts of unlabelled C, D2, D1 and D1m using 32P-C or 32P-D2, are indicated in each graph. Results are expressed as a percentage of the radioactivity bound in the absence of unlabelled oligonucleotides and plotted as a function of the log (ng oligonucleotide) in the reaction.
Mentions: Mammary gland nuclear extracts incubated with the 32P labelled double strand D2 probe (32P-D2, corresponding to the more distal potential STAT5 sequence), formed a complex that migrated at a position similar to that observed for the Consensus STAT5 probe, indicated as C in Figure 1B (Figure 6A). The intensity of the signals detected for both complexes was similar. Over 90% of both D2 and C complexes were supershifted with STAT5a and STAT5b antibodies, clearly indicating that these potential STAT5 binding sites interact with both the STAT5a and STAT5b present in the extract. Weaker signals of the same mobility were also observed with 32P-D1. They were partially supershifted (15%) by STAT5 antibodies (Figure 6A, lower panel). The signals for 32P-D1m were even weaker (Figure 6A). Increasing the amounts of nuclear extracts did not increase the intensity of signals observed with D1 or D1m complexes, thus demonstrating that the weak band shifts were observed under saturating conditions (data not shown). These weak band shift signals could be anticipated by reported analysis of STAT5-DNA binding specificity [35]. The methylation of the CpG within the D1 sequence at most only marginally interferes with this weak in vitro interaction.

Bottom Line: Once daily milking (ODM) induces a reduction in milk production when compared to twice daily milking (TDM).Our results therefore shed light on mechanisms that help to explain how milk production is almost, but not fully, restored when TDM is resumed (15.1 ± 0.2 kg/day instead of 16.2 ± 0.2 kg/day, p<0.01).We now need to clarify whether the transcription of this novel RNA is a consequence of STAT5 interacting with the CSN1S1 distal region, or whether it plays a role in the chromatin structure of this region.

View Article: PubMed Central - PubMed

Affiliation: INRA, UMR1313 Génétique Animale et Biologie Intégrative, Jouy-en-Josas, France.

ABSTRACT
Once daily milking (ODM) induces a reduction in milk production when compared to twice daily milking (TDM). Unilateral ODM of one udder half and TDM of the other half, enables the study of underlying mechanisms independently of inter-individual variability (same genetic background) and of environmental factors. Our results show that in first-calf heifers three CpG, located 10 kb upstream from the CSN1S1 gene were methylated to 33, 34 and 28%, respectively, after TDM but these levels were higher after ODM, 38, 38 and 33%, respectively. These methylation levels were much lower than those observed in the mammary gland during pregnancy (57, 59 and 50%, respectively) or in the liver (74, 78 and 61%, respectively). The methylation level of a fourth CpG (CpG4), located close by (29% during TDM) was not altered after ODM. CpG4 methylation reached 39.7% and 59.5%, during pregnancy or in the liver, respectively. CpG4 is located within a weak STAT5 binding element, arranged in tandem with a second high affinity STAT5 element. STAT5 binding is only marginally modulated by CpG4 methylation, but it may be altered by the methylation levels of the three other CpG nearby. Our results therefore shed light on mechanisms that help to explain how milk production is almost, but not fully, restored when TDM is resumed (15.1 ± 0.2 kg/day instead of 16.2 ± 0.2 kg/day, p<0.01). The STAT5 elements are 100 bp away from a region transcribed in the antisense orientation, in the mammary gland during lactation, but not during pregnancy or in other reproductive organs (ovary or testes). We now need to clarify whether the transcription of this novel RNA is a consequence of STAT5 interacting with the CSN1S1 distal region, or whether it plays a role in the chromatin structure of this region.

Show MeSH
Related in: MedlinePlus