Lam et al. 10.1073/pnas.0507947103. |
Supporting Figure 6
Supporting Figure 7
Supporting Figure 8
Supporting Methods
Fig. 6. Transcription occurs within human centromeric chromatin (CEN chromatin) RT-PCR of RNA immunoprecipitated with anti-CENP-A or anti-methylated H3 antibodies was used to detect blasticidin (bsr) transcripts on artificial chromosomes. (A) Bsr transcripts were associated with H3K4me3 and CENP-A, but not with H3K9me3. PAC109, artificial chromosome DNA input construct, was subjected to RT-PCR in the presence (+) or absence (–) of reverse transcriptase (RT). (B) Bands from five independent RT-PCR reactions were quantitated to show increased enrichment of bsr transcripts with euchromatic and centromeric, but not heterochromatic, histone modifications. Enrichment difference between CENP-A and H3K9me3 was determined to be statistically significant by using a Student t test (P = 0.03).
Fig. 7. Increased dosage of CENP-A in human cell lines using FLAG- tagged constructs. Percentage IP recovery (%IP) of CENP-A, H3K4me2, H3K9me2, and H3K9me3 over input DNA for a-satellite was determined in normal (HT1080, X4, and X5) and FLAG-CENP-A-overexpressing (HTFL1c1, X4FLCpa1.4, and X5FLCpa1.2) cell lines. (A) Nuclear extracts from each cell line were immunoblotted with anti-CENP-A and anti-FLAG antibodies and normalized against actin. (B) Densitometry analysis of endogenous CENP-A (black bars) and FLAG-CENP-A (gray bars) was normalized to the amount of actin. In each cell line, FLAG-CENP-A is overexpressed by 50% over endogenous levels.
Fig. 8. Chromatin organization at mammalian centromeres. Proposed model to describe the dynamic nature of chromatin domains within the human centromere region. Linear organization of centromeric histones is schematically represented, but the array of CENP-A and modified histones is thought to produce the three-dimensional structure of the kinetochore (10).(A) CEN chromatin is organized as interspersed blocks of CENP-A nucleosomes (red) and H3K4me2 nucleosomes (green). CEN chromatin is flanked by H3K9me2 nucleosomes (yellow). Nucleosomes containing H3K9me3 (brown) and H3-K27 methylation (gray) are also assembled at a-satellite but are located further away. (B) When CENP-A dosage is increased by 50%, CENP-A spreads in cis, replacing nucleosomes containing H3K9me2 but not H3K9me3. Heterochromatin defined by H3K9me2 may serve as a passive boundary between CEN chromatin and constitutive heterochromatin. Constitutive heterochromatin may be a stronger barrier that constrains CEN chromatin, in accordance with a proposed model in Drosophila (21).
Supporting Methods
SDS/PAGE and Western Blotting.
Equal amount of nuclear protein extracts were separated on a 15% SDS/polyacrylamide gel and transferred onto poly(vinylidene difluoride) membrane (Millipore). Antibodies to CENP-A (Abcam, Cambridge, MA), FLAG M2 (Sigma) and b-actin (Abcam) were detected by using ECL, and bands were quantitated and normalized to b-actin by densitometry.RNA-Chromatin Immunoprecipitation (RNA ChIP) and RT-PCR.
Native chromatin preparations were used as starting material in all RNA-ChIP experiments. RT-PCR was carried out using both Titan One-Step RT-PCR kit (Roche) and Superscript III One-Step kit (Invitrogen), with blasticidin primers. Bands in RT-PCR reactions containing reverse transcriptase (RT) were quantitated by densitometry and normalized to mock. RNA-IPs were performed from five independent experiments.