Identification of Chromatin proteins involved in gene repression in lamina-associated domains (RNA-Seq)

Lamina-associated domains (LADs) are large chromatin regions that are associated with the nuclear lamina and form a repressive environment for transcription. The molecular players that mediate gene repression in LADs are currently unknown. Here we performed a full-genome genetic screen in human cells using LAD-integrated fluorescent reporters to identify such regulators. Surprisingly, the screen identified very few lamina proteins, but revealed roles for dozens of known chromatin regulators. Among these are the negative elongation factor (NELF) complex and interacting factors, suggesting that regulation of RNA polymerase pausing can be a mechanism to repress transcription in LADs. Furthermore, the chromatin remodeler complex BAF and the activation complex Mediator can work both as activators and repressors in LADs, depending on the local context and possibly rewiring of heterochromatin. Our data suggest that fundamental regulatory steps of the transcription process and chromatin remodeling, rather than interaction with NL proteins, have a major role in the regulation of transcription in LADs. RNASEQ experiments were performed on a clonal HAP1 cell line genetically modified to express dCAS9-KRAB domain and transduced with a lentiguide against ADAM22 (pLB187,negative control as not expressed in HAP1) or lentiguides against NELFE or NELFB. RNA was extracted 144 hours following transduction.