TFIID Enables RNA Polymerase II Promoter-Proximal Pausing

Charli B Fant; Cecilia B Levandowski; Kapil  Gupta; Zachary L Maas; John  Moir; Jonathan D Rubin; Andrew  Sawyer; Meagan N Esbin; Jenna K Rimel; Olivia  Luyties; Michael T Marr; Imre Berger; Robin D Dowell; Dylan J Taatjes

doi:10.1016/j.molcel.2020.03.008

TFIID Enables RNA Polymerase II Promoter-Proximal Pausing

Charli B Fant, Cecilia B Levandowski, Kapil Gupta, Zachary L Maas, John Moir, Jonathan D Rubin, Andrew Sawyer, Meagan N Esbin, Jenna K Rimel, Olivia Luyties, Michael T Marr, Imre Berger, Robin D Dowell, Dylan J Taatjes^*

^*Corresponding author for this work

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Abstract

RNA polymerase II (RNAPII) transcription is governed by the pre-initiation complex (PIC), which contains TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH, RNAPII, and Mediator. After initiation, RNAPII enzymes pause after transcribing less than 100 bases; precisely how RNAPII pausing is enforced and regulated remains unclear. To address specific mechanistic questions, we reconstituted human RNAPII promoter-proximal pausing in vitro, entirely with purified factors (no extracts). As expected, NELF and DSIF increased pausing, and P-TEFb promoted pause release. Unexpectedly, the PIC alone was sufficient to reconstitute pausing, suggesting RNAPII pausing is an inherent PIC function. In agreement, pausing was lost upon replacement of the TFIID complex with TATA-binding protein (TBP), and PRO-seq experiments revealed widespread disruption of RNAPII pausing upon acute depletion (t = 60 min) of TFIID subunits in human or Drosophila cells. These results establish a TFIID requirement for RNAPII pausing and suggest pause regulatory factors may function directly or indirectly through TFIID

Original language	English
Pages (from-to)	785-793.e8
Number of pages	18
Journal	Molecular Cell
Volume	78
Issue number	4
Early online date	30 Mar 2020
DOIs	https://doi.org/10.1016/j.molcel.2020.03.008
Publication status	Published - 21 May 2020

Keywords

TFIID
TRIM-Away
TAF1
RNA polymerase II
pausing
NELF
DSIF
TBP
P-TEFb
PRO-seq

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title = "TFIID Enables RNA Polymerase II Promoter-Proximal Pausing",

abstract = "RNA polymerase II (RNAPII) transcription is governed by the pre-initiation complex (PIC), which contains TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH, RNAPII, and Mediator. After initiation, RNAPII enzymes pause after transcribing less than 100 bases; precisely how RNAPII pausing is enforced and regulated remains unclear. To address specific mechanistic questions, we reconstituted human RNAPII promoter-proximal pausing in vitro, entirely with purified factors (no extracts). As expected, NELF and DSIF increased pausing, and P-TEFb promoted pause release. Unexpectedly, the PIC alone was sufficient to reconstitute pausing, suggesting RNAPII pausing is an inherent PIC function. In agreement, pausing was lost upon replacement of the TFIID complex with TATA-binding protein (TBP), and PRO-seq experiments revealed widespread disruption of RNAPII pausing upon acute depletion (t = 60 min) of TFIID subunits in human or Drosophila cells. These results establish a TFIID requirement for RNAPII pausing and suggest pause regulatory factors may function directly or indirectly through TFIID",

keywords = "TFIID, TRIM-Away, TAF1, RNA polymerase II, pausing, NELF, DSIF, TBP, P-TEFb, PRO-seq",

author = "Fant, {Charli B} and Levandowski, {Cecilia B} and Kapil Gupta and Maas, {Zachary L} and John Moir and Rubin, {Jonathan D} and Andrew Sawyer and Esbin, {Meagan N} and Rimel, {Jenna K} and Olivia Luyties and Marr, {Michael T} and Imre Berger and Dowell, {Robin D} and Taatjes, {Dylan J}",

year = "2020",

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doi = "10.1016/j.molcel.2020.03.008",

language = "English",

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journal = "Molecular Cell",

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TY - JOUR

T1 - TFIID Enables RNA Polymerase II Promoter-Proximal Pausing

AU - Fant, Charli B

AU - Levandowski, Cecilia B

AU - Gupta, Kapil

AU - Maas, Zachary L

AU - Moir, John

AU - Rubin, Jonathan D

AU - Sawyer, Andrew

AU - Esbin, Meagan N

AU - Rimel, Jenna K

AU - Luyties, Olivia

AU - Marr, Michael T

AU - Berger, Imre

AU - Dowell, Robin D

AU - Taatjes, Dylan J

PY - 2020/5/21

Y1 - 2020/5/21

N2 - RNA polymerase II (RNAPII) transcription is governed by the pre-initiation complex (PIC), which contains TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH, RNAPII, and Mediator. After initiation, RNAPII enzymes pause after transcribing less than 100 bases; precisely how RNAPII pausing is enforced and regulated remains unclear. To address specific mechanistic questions, we reconstituted human RNAPII promoter-proximal pausing in vitro, entirely with purified factors (no extracts). As expected, NELF and DSIF increased pausing, and P-TEFb promoted pause release. Unexpectedly, the PIC alone was sufficient to reconstitute pausing, suggesting RNAPII pausing is an inherent PIC function. In agreement, pausing was lost upon replacement of the TFIID complex with TATA-binding protein (TBP), and PRO-seq experiments revealed widespread disruption of RNAPII pausing upon acute depletion (t = 60 min) of TFIID subunits in human or Drosophila cells. These results establish a TFIID requirement for RNAPII pausing and suggest pause regulatory factors may function directly or indirectly through TFIID

AB - RNA polymerase II (RNAPII) transcription is governed by the pre-initiation complex (PIC), which contains TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH, RNAPII, and Mediator. After initiation, RNAPII enzymes pause after transcribing less than 100 bases; precisely how RNAPII pausing is enforced and regulated remains unclear. To address specific mechanistic questions, we reconstituted human RNAPII promoter-proximal pausing in vitro, entirely with purified factors (no extracts). As expected, NELF and DSIF increased pausing, and P-TEFb promoted pause release. Unexpectedly, the PIC alone was sufficient to reconstitute pausing, suggesting RNAPII pausing is an inherent PIC function. In agreement, pausing was lost upon replacement of the TFIID complex with TATA-binding protein (TBP), and PRO-seq experiments revealed widespread disruption of RNAPII pausing upon acute depletion (t = 60 min) of TFIID subunits in human or Drosophila cells. These results establish a TFIID requirement for RNAPII pausing and suggest pause regulatory factors may function directly or indirectly through TFIID

KW - TFIID

KW - TRIM-Away

KW - TAF1

KW - RNA polymerase II

KW - pausing

KW - NELF

KW - DSIF

KW - TBP

KW - P-TEFb

KW - PRO-seq

U2 - 10.1016/j.molcel.2020.03.008

DO - 10.1016/j.molcel.2020.03.008

M3 - Editorial (Academic Journal)

C2 - 32229306

SN - 1097-2765

VL - 78

SP - 785-793.e8

JO - Molecular Cell

JF - Molecular Cell

IS - 4

ER -

TFIID Enables RNA Polymerase II Promoter-Proximal Pausing

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