The arguably most fun thing about science is when your supervisor tells you to just do Experiment X to test hypothesis, but then they kind of forget to tell you how complicated the techniques are to perform that experiment, not to mention all the optimization you would need to do. I personally have never done a chromatin immunoprecipitation (ChIP), and since I wasn’t in genomics, the most sequencing I ever did was setting up quick reactions for the core facility to tell me that my gene constructs were correctly built. ChIP does sound rather simple when explained in class, but when you read up on the protocols,¹ there are some limitations to what ChIP can do, especially given the large amount of starting material you need for the typical experiment. Luckily, in recent years, scientists have started to use an alternative technique called Cleavage Under Targets and Tagmentation, or CUT&Tag, which ABclonal is pleased to support through our antibody reagents.

Large-scale sequencing projects have great potential to provide a wealth of knowledge to scientists and the public. Perhaps the most celebrated project of this nature is the Human Genome Project (HGP) which was completed in 2003. For many, the multi-billion endeavor was considered a “moonshot” for biology, but with its successful completion (99% of the euchromatic genome sequenced with 99.99% accuracy) came the launch of many other large-scale sequencing projects such as the Cancer Genome Atlas (2005) or more recently, the Earth Bio-genome Project (2018). The introduction of large-scale quantitative methods, such as next-generation sequencing, have also made these projects feasible.

The G1/S cell cycle checkpoints control whether eukaryotic cells enter the S phase (synthesis phase) of DNA synthesis after having properly completed the G1 phase to ensure the cell has enough energy and resources to begin DNA replication. Two cell cycle kinase complexes, CDK4/6-Cyclin D and CDK2- Cyclin E, work together to relieve the inhibition of dynamic transcriptional complexes containing retinoblastoma protein (Rb) and E2F. In cells undefined during the G1 phase, hypophosphorylated Rb binds to the E2F-DP1 transcription factor and forms an inhibitory complex with HDAC, thereby inhibiting downstream key transcriptional activities. Clear entry into the S phase is achieved by continuous phosphorylation of Rb by Cyclin D-CDK4/6 and Cyclin E-CDK2, which separates the transcription factor E2F from the inhibitory complex and allows transcription of the gene required for DNA replication. After the growth factor disappears, the expression level of cyclin D is down-regulated by down-regulation of protein expression and phosphorylation-dependent degradation. Without a proper G1/S checkpoint, the cell could arrest or potentially undergo aberrant processes that could lead to disease states such as cancer.

Proteins known as transcription factors play a crucial role in gene regulation by activating, enhancing, and even silencing a gene’s expression.  Many textbooks and resources compare transcription factors (TFs) to something like an on/off switch for gene transcription. However, it is a bit more complicated than just turning gene expression on or off. Various properties (e.g. binding affinity, specificity, and genetic variance of binding sites) impact the binding of TFs to DNA, thereby altering gene expression. To study transcription and how it is regulated, scientists study TF-DNA interactions on a genome-wide level.