9:10a – 8:00p, 9:50p – 2:30a

Ph project

revising manuscript draft

• working on abstract
• refocusing discussion of PcG clusters. Need to distinguish between the sparse micron-scale PcG bodies other people have studied and the numerous, nano-scale bodies we focus on.

Notes on Ph project organization / motivation

previous work has divided PcG organization into two structural categories: PcG bodies, and dispersed nuclear signal.

It is not entirely evident the large bodies visible by conventional microscopy are relevant to gene regulation:
1. For example, many (most?) Pc target genes are found outside of these bodies in a large fraction of cells.
2. These bodies disappear in hypertonic solution, but DAPI dense and H3K27me3 dense domains remain intact. (and presumably so does silencing?)

Our work demonstrates that much of this distinction is a consequence of technical limitations of conventional microscopy, rather than a fundamental difference in organization: PcG proteins are organized in bodies throughout the nucleus. This cluster size is power-law distributed, and only the largest clusters are distinguishable to conventional imaging methods — the small ones just blur together.

We propose that clustering is an important part of PcG repressive activity, and that it is mediated in part through the SAM domain of Ph. This clustering is important not just for the formation of large PcG “bodies”, but for clusters of PcG proteins at a whole range of scales, which function in efficient silencing of target genes.
Additionally we find clustering independent loci … (are these also silenced? e.g. does expression of Ph clustering independent genes change in PcG knockdown or Ph mutant backgrounds?)

Ph data analysis

• just need more data on size distribution of bodies. Can get some decent data from the 647 Ph imaging in the respective Ph and PhWt backgrounds (even though the 750 didn’t work well). Imaging of 647 flag indicates high transfection rate (just variable levels). So we should just be able to use nuclei from the slide straight. Be
• Starting writing ClusterStats function for analysis of Ph data. Each localization gets assigned a cluster and we report that cluster’s area

Team project / mentoring

• reviewing slides for Hao’s group meeting, 11a-2:30p

Chromatin Project

Cell staining

• Finish hybridizations for F03+F04 and G01+G02 cells.

STORM

• start imaging F03 + F04 as a contiguous ~200 kb region of BX-C
• dots seem spread out. These cells were also fixed a while ago (~1/6), I think we shouldn’t keep them this long.
• plan to repeat staining with fresh fixed cells tomorrow